WorldWideScience

Sample records for lymphoblastoid lines derived

  1. Differences between the genomes of lymphoblastoid cell lines and blood-derived samples

    Directory of Open Access Journals (Sweden)

    Joesch-Cohen LM

    2017-02-01

    Full Text Available Lena M Joesch-Cohen, Gustavo Glusman Institute for Systems Biology, Seattle, WA, USA Abstract: Lymphoblastoid cell lines (LCLs represent a convenient research tool for expanding the amount of biologic material available from an individual. LCLs are commonly used as reference materials, most notably from the Genome in a Bottle Consortium. However, the question remains how faithfully LCL-derived genome assemblies represent the germline genome of the donor individual as compared to the genome assemblies derived from peripheral blood mononuclear cells. We present an in-depth comparison of a large collection of LCL- and peripheral blood mononuclear cell-derived genomes in terms of distributions of coverage and copy number alterations. We found significant differences in the depth of coverage and copy number calls, which may be driven by differential replication timing. Importantly, these copy number changes preferentially affect regions closer to genes and with higher GC content. This suggests that genomic studies based on LCLs may display locus-specific biases, and that conclusions based on analysis of depth of coverage and copy number variation may require further scrutiny. Keywords: genomics, whole-genome sequencing, viral transformation, copy number changes, bioinformatics

  2. Porcine circovirus type 2 morphogenesis in a clone derived from the l35 lymphoblastoid cell line.

    Science.gov (United States)

    Rodríguez-Cariño, C; Duffy, C; Sánchez-Chardi, A; McNeilly, F; Allan, G M; Segalés, J

    2011-01-01

    Porcine circovirus type 2 (PCV2) is the essential infectious agent of post-weaning multisystemic wasting syndrome (PMWS), one of the most important diseases of swine. Although several studies have described different biological properties of the virus, some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe for the first time a complete morphogenesis study of PCV2 in a clone of the lymphoblastoid L35 cell line at the ultrastructural level using electron microscopy techniques. Cells were infected with PCV2 at a multiplicity of infection of 10 and examined at 0, 6, 12, 24, 48, 60 and 72h post-infection. PCV2 was internalized by endocytosis, after which the virus aggregated in intracytoplasmic inclusion bodies (ICIs). Subsequently, PCV2 was closely associated with mitochondria, completing a first cytoplasmic phase. The virus entered the nucleus for replication and virus assembly and encapsidation occurred with the participation of the nuclear membrane. Immature virions left the nucleus and formed ICIs in a second cytoplasmic phase. The results suggest that at the end of the replication cycle (between 24 and 48h), PCV2 was released either by budding of mature virion clusters or by lysis of apoptotic or dead cells. In conclusion, the L35-derived clone represents a suitable in-vitro model for PCV2 morphogenesis studies and characterization of the PCV2 replication cycle.

  3. File list: Pol.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306580,SRX306578,SRX306576,SRX306575 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  4. File list: His.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...5,SRX306570,SRX106080,SRX306566,SRX306568 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  5. File list: ALL.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell line...ve.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  6. File list: Oth.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  7. File list: Oth.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  8. File list: His.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...2,SRX306570,SRX356718,SRX356754,SRX026069 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  9. File list: DNS.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...091606,SRX469953,SRX091596 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  10. File list: Unc.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  11. File list: Pol.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306580,SRX306575,SRX306578,SRX306576 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  12. File list: Unc.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  13. File list: His.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...7,SRX356718,SRX356754,SRX026054,SRX026069 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  14. File list: Unc.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  15. File list: His.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...8,SRX356735,SRX356754,SRX306535,SRX026069 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  16. File list: DNS.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...8,SRX091598,SRX091626,SRX469951,SRX469953,SRX469955 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  17. File list: Pol.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306575,SRX306580,SRX306576,SRX306578 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  18. File list: DNS.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...0,SRX091618,SRX091595,SRX091598,SRX469953 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  19. File list: Unc.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  20. File list: Pol.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306575,SRX306580,SRX306576,SRX306578 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  1. File list: DNS.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...8,SRX091623,SRX091605,SRX469953,SRX469951,SRX469955 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  2. File list: ALL.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell line...://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  3. File list: ALL.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell line...://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  4. File list: Oth.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  5. File list: ALL.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell line...6,SRX306568,SRX469953,SRX144527,SRX144520,SRX091596 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  6. File list: Oth.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  7. Utilization of Lymphoblastoid Cell Lines as a System for the Molecular Modeling of Autism

    Science.gov (United States)

    Baron, Colin A.; Liu, Stephenie Y.; Hicks, Chindo; Gregg, Jeffrey P.

    2006-01-01

    In order to provide an alternative approach for understanding the biology and genetics of autism, we performed statistical analysis of gene expression profiles of lymphoblastoid cell lines derived from children with autism and their families. The goal was to assess the feasibility of using this model in identifying autism-associated genes.…

  8. Characterization of a lymphoblastoid line deleted for lambda immunoglobulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Hough, C.A., White, B.N., Holden, J.A. [Queen`s Univ., Ontario (Canada)

    1995-04-01

    While characterizing the cat eye syndrome (CES) supernumerary chromosome for the presence of {lambda} immunoglobulin gene region sequences, a lymphoblastoid cell line from one CES patient was identified in which there was selection of cells deleted from some IGLC and IGLV genes. Two distinct deletions, one on each chromosome 22, were identified, presumably arising from independent somatic recombination events occurring during B-lymphocyte differentiation. The extent of the deleted regions was determined using probes from the various IGLV subgroups and they each covered at least 82 kilobases. The precise definition of the deletions was not possible because of conservation of some restriction sites in the IGLV region. The cell line was used to map putative IGLV genes within the recombinant phage {lambda}V{lambda}135 to the distal part of the IGLV gene region. 35 refs., 4 figs.

  9. File list: NoD.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 No description Blood Lymphoblastoid cell line...,SRX1027609 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  10. File list: InP.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 Input control Blood Lymphoblastoid cell line...osciencedbc.jp/kyushu-u/hg19/assembled/InP.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  11. File list: NoD.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 No description Blood Lymphoblastoid cell line...,SRX1027609 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  12. File list: NoD.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 No description Blood Lymphoblastoid cell line...,SRX1027609 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  13. File list: NoD.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 No description Blood Lymphoblastoid cell line...,SRX1027609 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  14. File list: InP.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 Input control Blood Lymphoblastoid cell line...osciencedbc.jp/kyushu-u/hg19/assembled/InP.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  15. File list: InP.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 Input control Blood Lymphoblastoid cell line...osciencedbc.jp/kyushu-u/hg19/assembled/InP.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  16. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Hwang, Meeyul [Research Center for Biomedical Resource of Oriental Medicine, Daegu Haany University (Korea, Republic of); Kim, Ji-Hyun [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Han, Bok-Ghee, E-mail: bokghee@nih.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Jeon, Jae-Pil, E-mail: jpjeon@cdc.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  17. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of); Jeon, Jae-Pil, E-mail: jaepiljeon@hanmail.net [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)

    2013-11-15

    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  18. Human iPSC-derived neurons and lymphoblastoid cells for personalized medicine research in neuropsychiatric disorders

    Science.gov (United States)

    Gurwitz, David

    2016-01-01

    The development and clinical implementation of personalized medicine crucially depends on the availability of high-quality human biosamples; animal models, although capable of modeling complex human diseases, cannot reflect the large variation in the human genome, epigenome, transcriptome, proteome, and metabolome. Although the biosamples available from public biobanks that store human tissues and cells may represent the large human diversity for most diseases, these samples are not always sufficient for developing biomarkers for patient-tailored therapies for neuropsychiatric disorders. Postmortem human tissues are available from many biobanks; nevertheless, collections of neuronal human cells from large patient cohorts representing the human diversity remain scarce. Two tools are gaining popularity for personalized medicine research on neuropsychiatric disorders: human induced pluripotent stem cell-derived neurons and human lymphoblastoid cell lines. This review examines and contrasts the advantages and limitations of each tool for personalized medicine research.

  19. Human lymphoblastoid B-cell lines reprogrammed to EBV-free induced pluripotent stem cells.

    Science.gov (United States)

    Rajesh, Deepika; Dickerson, Sarah J; Yu, Junying; Brown, Matthew E; Thomson, James A; Seay, Nicholas J

    2011-08-18

    Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations. We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hematopoietic, cardiac, neural, and hepatocyte-like lineages. Significantly, although the parental LCLs express viral EBNA-1 and other Epstein-Barr virus latency-related elements for their survival, their presence was not detectable in LCL-iPSCs. Thus, reprogramming LCLs could offer an unlimited source for patient-specific iPSCs.

  20. Protein degradation in a LAMP-2-deficient B-lymphoblastoid cell line from a patient with Danon disease.

    Science.gov (United States)

    Sánchez-Lanzas, Raul; Alvarez-Castelao, Beatriz; Bermejo, Teresa; Ayuso, Teresa; Tuñón, Teresa; Castaño, José G

    2016-08-01

    Danon disease, a condition characterized by cardiomyopathy, myopathy, and intellectual disability, is caused by mutations in the LAMP-2 gene. Lamp-2A protein, generated by alternative splicing from the Lamp-2 pre-mRNA, is reported to be the lysosomal membrane receptor essential for the chaperone-mediated autophagic pathway (CMA) aimed to selective protein targeting and translocation into the lysosomal lumen for degradation. To study the relevance of Lamp-2 in protein degradation, a lymphoblastoid cell line was obtained by EBV transformation of B-cells from a Danon patient. The derived cell line showed no significant expression of Lamp-2 protein. The steady-state mRNA and protein levels of alpha-synuclein, IΚBα, Rcan1, and glyceraldehyde-3-phosphate dehydrogenase, four proteins reported to be selective substrates of the CMA pathway, were similar in control and Lamp-2-deficient cells. Inhibition of protein synthesis showed that the half-life of alpha-synuclein, IΚBα, and Rcan1 was similar in control and Lamp-2-deficient cells, and its degradation prevented by proteasome inhibitors. Both in control and Lamp-2-deficient cells, induction of CMA and macroautophagy by serum and aminoacid starvation of cells for 8h produced a similar decrease in IΚBα and Rcan1 protein levels and was prevented by the addition of lysosome and autophagy inhibitors. In conclusion, the results presented here showed that Lamp-2 deficiency in human lymphoblastoid cells did not modify the steady-state levels or the degradation of several protein substrates reported as selective substrates of the CMA pathway.

  1. Cellular models for mitochondrial DNA-based diseases: lymphoblastoid cell lines and transmitochondrial cybrids.

    Science.gov (United States)

    Jiji, Sun; Xiaoxu, Zhao; Lihua, Qiao; Shuang, Mei; Zhipeng, Nie; Qinghai, Zhang; Yanchun, Ji; Pingping, Jiang; Min-Xin, Guan

    2016-07-20

    Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial DNA-based diseases which have been studied using Lymphoblastoid cell lines (LCLs) and transmitochondrial cybrids. Individual genetic information is preserved permanently in LCLs while the development of transmitochondrial cybrids provide ex-vivo cellular platform to study molecular mechanism of mitochondrial DNA-based diseases. The cytoplasmic donor cells for previous transmitochondrial cybrids come from patient's tissue or platelet directly. Here, we depicted in details the principle, methods and techniques to establish LCLs from frozen peripheral bloods harboring mitochondrial 4401G > A mutation by infection of Epstein Barr virus, and then to generate cybrids using ρ(0) 206 and LCLs. The process of establishing these two cellular models was summarized into four steps as follows: (1) Generation of LCLs; (2) Transformation; (3) Selection; (4) Verification. To faithfully represent the function of mtDNA mutation, we analyzed and identified the sites of mtDNA mutations and copy numbers of each cellular models as well as the karyotype of transmitochondrial cybrids. Those clones with consistent parameters were selected for preservation and future analysis of the function of point mutations of mtDNA. Although these two cellular models play important roles in understanding molecular mechanism of mitochondrial DNA-based diseases on the cellular level, their limitations should be considered when elucidating the character of tissue specificity of mitochondrial DNA-based diseases.

  2. Genotype instability during long-term subculture of lymphoblastoid cell lines.

    Science.gov (United States)

    Oh, Ji Hee; Kim, Young Jin; Moon, Sanghoon; Nam, Hye-Young; Jeon, Jae-Pil; Lee, Jong Ho; Lee, Jong-Young; Cho, Yoon Shin

    2013-01-01

    Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) promise to address the challenge posed by the limited availability of primary cells needed as a source of genomic DNA for genetic studies. However, the genetic stability of LCLs following prolonged culture has never been rigorously investigated. To evaluate genotypic errors caused by EBV integration into human chromosomes, we isolated genomic DNA from human peripheral blood mononuclear cells and LCLs collected from 20 individuals and genotyped the DNA samples using the Affymetrix 500K SNP array set. Genotype concordance measurements between two sources of DNA from the same individual indicated that genotypic discordance is negligible in early-passage LCLs (50 passages). Analysis of concordance on a chromosome-by-chromosome basis identified genomic regions with a high frequency of genotypic errors resulting from the loss of heterozygosity observed in late-passage LCLs. Our findings suggest that, although LCLs harvested during early stages of propagation are a reliable source of genomic DNA for genetic studies, investigations that involve genotyping of the entire genome should not use DNA from late-passage LCLs.

  3. In vitro cytotoxicity against Marek's disease lymphoblastoid cell lines after enzymatic removal of Marek's disease tumor-associated surface antigen.

    OpenAIRE

    Schat, K A; Murthy, K K

    1980-01-01

    Marek's disease tumor-associated surface antigen (MATSA) has been claimed to be the target of cytotoxic lymphocytes in in vitro tests for Marek's disease immunity. Treatment with papain, but not with trypsin or mixed glycosidases, removed MATSA from certain Marek's disease lymphoblastoid cell lines. Tumor cells with and without MATSA were used as target cells for in vitro studies on cell-mediated immune responses with sensitized spleen cells in a chromium release assay. The removal of MATSA d...

  4. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S. [Institute of Genetics, Taipei (Taiwan, Province of China)

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  5. Genetic analysis of human traits in vitro: drug response and gene expression in lymphoblastoid cell lines.

    Directory of Open Access Journals (Sweden)

    Edwin Choy

    2008-11-01

    Full Text Available Lymphoblastoid cell lines (LCLs, originally collected as renewable sources of DNA, are now being used as a model system to study genotype-phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance--i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.

  6. Linking short tandem repeat polymorphisms with cytosine modifications in human lymphoblastoid cell lines.

    Science.gov (United States)

    Zhang, Zhou; Zheng, Yinan; Zhang, Xu; Liu, Cong; Joyce, Brian Thomas; Kibbe, Warren A; Hou, Lifang; Zhang, Wei

    2016-02-01

    Inter-individual variation in cytosine modifications has been linked to complex traits in humans. Cytosine modification variation is partially controlled by single nucleotide polymorphisms (SNPs), known as modified cytosine quantitative trait loci (mQTL). However, little is known about the role of short tandem repeat polymorphisms (STRPs), a class of structural genetic variants, in regulating cytosine modifications. Utilizing the published data on the International HapMap Project lymphoblastoid cell lines (LCLs), we assessed the relationships between 721 STRPs and the modification levels of 283,540 autosomal CpG sites. Our findings suggest that, in contrast to the predominant cis-acting mode for SNP-based mQTL, STRPs are associated with cytosine modification levels in both cis-acting (local) and trans-acting (distant) modes. In local scans within the ±1 Mb windows of target CpGs, 21, 9, and 21 cis-acting STRP-based mQTL were detected in CEU (Caucasian residents from Utah, USA), YRI (Yoruba people from Ibadan, Nigeria), and the combined samples, respectively. In contrast, 139,420, 76,817, and 121,866 trans-acting STRP-based mQTL were identified in CEU, YRI, and the combined samples, respectively. A substantial proportion of CpG sites detected with local STRP-based mQTL were not associated with SNP-based mQTL, suggesting that STRPs represent an independent class of mQTL. Functionally, genetic variants neighboring CpG-associated STRPs are enriched with genome-wide association study (GWAS) loci for a variety of complex traits and diseases, including cancers, based on the National Human Genome Research Institute (NHGRI) GWAS Catalog. Therefore, elucidating these STRP-based mQTL in addition to SNP-based mQTL can provide novel insights into the genetic architectures of complex traits.

  7. Distinct lithium-induced gene expression effects in lymphoblastoid cell lines from patients with bipolar disorder.

    Science.gov (United States)

    Fries, Gabriel R; Colpo, Gabriela D; Monroy-Jaramillo, Nancy; Zhao, Junfei; Zhao, Zhongming; Arnold, Jodi G; Bowden, Charles L; Walss-Bass, Consuelo

    2017-09-19

    Lithium is the most commonly prescribed medication for the treatment of bipolar disorder (BD), yet the mechanisms underlying its beneficial effects are still unclear. We aimed to compare the effects of lithium treatment in lymphoblastoid cell lines (LCLs) from BD patients and controls. LCLs were generated from sixty-two BD patients (based on DSM-IV) and seventeen healthy controls matched for age, sex, and ethnicity. Patients were recruited from outpatient clinics from February 2012 to October 2014. LCLs were treated with 1mM lithium for 7 days followed by microarray gene expression assay and validation by real-time quantitative PCR. Baseline differences between groups, as well as differences between vehicle- and lithium-treated cells within each group were analyzed. The biological significance of differentially expressed genes was examined by pathway enrichment analysis. No significant differences in baseline gene expression (adjusted p-value Lithium treatment of LCLs from controls did not lead to any significant differences. However, lithium altered the expression of 236 genes in LCLs from patients; those genes were enriched for signaling pathways related to apoptosis. Among those genes, the alterations in the expression of PIK3CG, SERP1 and UPP1 were validated by real-time PCR. A significant correlation was also found between circadian functioning and CEBPG and FGF2 expression levels. In summary, our results suggest that lithium treatment induces expression changes in genes associated with the apoptosis pathway in BD LCLs. The more pronounced effects of lithium in patients compared to controls suggest a disease-specific effect of this drug. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  8. Individual radiosensitivity does not correlate with radiation-induced apoptosis in lymphoblastoid cell lines or CD{sup 3+} lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wistop, A.; Keller, U.; Grabenbauer, G.G.; Sauer, R.; Distel, L.V.R. [Dept. of Radiation Oncology, Friedrich Alexander Univ. Erlangen-Nuremberg, Erlangen (Germany); Sprung, C.N. [Div. of Research, Peter MacCallum Cancer Centre, East Melbourne, VIC (Australia)

    2005-05-01

    Background and purpose: spontaneous and radiation-induced apoptosis of lymphoblastoid cell lines (LCLs) derived from healthy donors, cancer patients and donors with radiosensitivity syndromes as well as CD{sup 3+} lymphocytes from patients with {>=} grade 3 late toxicity were investigated as a possible marker for the detection of individual radiosensitivity. These investigations are based on the hypothesis that hypersensitive patients have reduced levels of apoptosis after in vitro irradiation as a result of a defect in the signaling pathway. Material and methods: Epstein-Barr virus-(EBV-)transformed LCLs derived from five healthy donors, seven patients with heterozygous or homozygous genotype for ataxia-telangiectasia or Nijmegen breakage syndrome and five patients with {>=} grade 3 late toxicity (RTOG) were investigated. In addition, CD{sup 3+} lymphocytes from 21 healthy individuals and 18 cancer patients including five patients with a proven cellular hypersensitivity to radiation were analyzed. Cells were irradiated in vitro with a dose of 2 and 5 Gy and were incubated for 48 h. Apoptotic rates were measured by the TUNEL assay followed by customized image analysis. Results: four out of seven radiosensitivity syndrome patients were identified to have an increased cellular radiosensitivity as determined by reduced apoptotic rates after irradiation of their respective LCLs. Comparatively, only two of the five hypersensitive cancer patients were clearly identified by reduced apoptotic rates. Spontaneous apoptotic rates were very homogeneous among all 39 samples from controls and patients, while lymphocytes of all cancer patients showed significantly lower radiation-induced rates. Conclusion: only a subgroup of hypersensitive patients may be identified by reduction of radiation-induced apoptotic rate. It is concluded that the hypothesis according to which hypersensitive cells have reduced levels of apoptosis is only conditionally true. The authors suggest that this

  9. The Lymphoblastoid Cell Lines of Recurrence Condyloma Acuminatum Patients Produce Lower Level of Tumor Necrosis Factor Stimulated with LPS

    Institute of Scientific and Technical Information of China (English)

    刘冬先; 周礼义; 陈兴平

    2003-01-01

    To study the mechanism of Condyloma acuminatum (CA) recurrence, and the associa-tion of CA recurrence with the ability of the host derived lymphoblastoid cell lines (LCL) stimulatedby LPS to produce tumor necrosis factor (TNF), EBV-transformedB LCL were used as TNF pro-ducing cells. The ability of LCL stimulated by LPS to produce TNF was measured by bioassay.The results showed that the LCL from CA patients (including recurrent and non-recurrent CA pa-tients) produced similar level of TNF stimulated by LPS to that of normal controls (29.54% ±11.28% vs 34. 31%±11.46%, P=0. 1498). The LCL of CA recurrent patients produced significantly lower amount of TNF than that of non-recurrent CA patients (23.72%±7.41% vs 37.33%± 11.10%, P=0. 0032). Compared with the normal controls, CA recurrent patients showed a de-creased ability to produce TNF (23.72% ± 7.41 vs 34.31% ± 11.46, P = 0. 0054 ), whereas CAnon recurrent patients had the similar ability to the controls (37.33%±11.10 vs 34.31%±11.46,P=0. 4914). It was concluded that the onset of CA was not relevant to the individual's ability toproduce TNF. But the recurrence of CA was associated with the ability to produce TNF. It was al-so indicated that the TNF involved cellular immunity might play an important role in the clearanceof the residual HPV by the host after treatment.

  10. Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte;

    2013-01-01

    /short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA....

  11. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain

    OpenAIRE

    Nguyen, Anhthu; Rauch, Tibor A; Pfeifer, Gerd P.; Hu, Valerie W.

    2010-01-01

    Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well ...

  12. Clonal analysis of a human lymphoblastoid cell line (B17) secreting antibody to N-acetyl-D-glucosamine.

    Science.gov (United States)

    Polke, C; Greger, B; Steinitz, M; Eichmann, K

    1982-10-01

    In this paper we analyse the clonal composition of a human lymphoblastoid B-cell line secreting IgM/k antibody to N-acetyl-D-glucosamine, the immunodominant sugar of Group-A-streptococcal carbohydrate. Besides non-antibody secreting cells, the line consists of two clonotypes of antibody-secreting cells: B17 cells producing over 90% and F6 cells producing less than 10% of the antibody in the supernatant. The proportions of B17 and F6 cells in the cell line seem to be similar to the proportion of antibodies in the supernatant. F6 cells can be isolated by cloning and maintained as stable lines, whereas this is more difficult with B17 cells. The results suggest that upon establishment of the line, at least two N-acetyl-D-glucosamine-specific B cells were immortalized and coexist together as independent clonotypes. Although F6 cells seem to have a slight tissue culture advantage, they represent the minor clonotype in the B17 cell line.

  13. In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

    Science.gov (United States)

    Mohamed, Yehia S; Dunnion, Debbie; Teobald, Iryna; Walewska, Renata; Browning, Michael J

    2012-10-12

    Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Genome-wide association study for biomarker identification of Rapamycin and Everolimus using a lymphoblastoid cell line system

    Directory of Open Access Journals (Sweden)

    Jing eJiang

    2013-08-01

    Full Text Available The mammalian target of rapamycin (mTOR inhibitors, a set of promising potential anti-cancer agents, has shown response variability among individuals. This study aimed to identify novel biomarkers and mechanisms that might influence the response to Rapamycin and Everolimus. Genome-wide association (GWA analyses involving single nucleotide polymorphisms (SNPs, mRNA and microRNAs microarray data were assessed for association with area under the cytotoxicity dose response curve (AUC of two mTOR inhibitors in 272 human lymphoblastoid cell lines (LCLs. Integrated analysis among SNPs, expression data, microRNA data and AUC values were also performed to help select candidate genes for further functional characterization. Functional validation of candidate genes using siRNA screening in multiple cell lines followed by MTS assays for the two mTOR inhibitors were performed. We found that 16 expression probe sets (genes that overlapped between the two drugs were associated with AUC values of two mTOR inhibitors. 127 and 100 SNPs had P<10-4, while 8 and 10 SNPs had P<10-5 with Rapamycin and Everolimus AUC, respectively. Functional studies indicated that 13 genes significantly altered cell sensitivity to either one or both drugs in at least one cell line. Additionally, one microRNA, miR-10a, was significantly associated with AUC values for both drugs and was shown to repress expression of genes that were associated with AUC and desensitize cells to both drugs. In summary, this study identified genes and a microRNA that might contribute to response to mTOR inhibitors.

  15. Molecular Signatures of Cardiac Defects in Down Syndrome Lymphoblastoid Cell Lines Suggest Altered Ciliome and Hedgehog Pathways

    Science.gov (United States)

    Ripoll, Clémentine; Rivals, Isabelle; Ait Yahya-Graison, Emilie; Dauphinot, Luce; Paly, Evelyne; Mircher, Clothilde; Ravel, Aimé; Grattau, Yann; Bléhaut, Henri; Mégarbane, André; Dembour, Guy; de Fréminville, Bénédicte; Touraine, Renaud; Créau, Nicole; Potier, Marie Claude; Delabar, Jean Maurice

    2012-01-01

    Forty percent of people with Down syndrome exhibit heart defects, most often an atrioventricular septal defect (AVSD) and less frequently a ventricular septal defect (VSD) or atrial septal defect (ASD). Lymphoblastoid cell lines (LCLs) were established from lymphocytes of individuals with trisomy 21, the chromosomal abnormality causing Down syndrome. Gene expression profiles generated from DNA microarrays of LCLs from individuals without heart defects (CHD−; n = 22) were compared with those of LCLs from patients with cardiac malformations (CHD+; n = 21). After quantile normalization, principal component analysis revealed that AVSD carriers could be distinguished from a combined group of ASD or VSD (ASD+VSD) carriers. From 9,758 expressed genes, we identified 889 and 1,016 genes differentially expressed between CHD− and AVSD and CHD− and ASD+VSD, respectively, with only 119 genes in common. A specific chromosomal enrichment was found in each group of affected genes. Among the differentially expressed genes, more than 65% are expressed in human or mouse fetal heart tissues (GEO dataset). Additional LCLs from new groups of AVSD and ASD+VSD patients were analyzed by quantitative PCR; observed expression ratios were similar to microarray results. Analysis of GO categories revealed enrichment of genes from pathways regulating clathrin-mediated endocytosis in patients with AVSD and of genes involved in semaphorin-plexin-driven cardiogenesis and the formation of cytoplasmic microtubules in patients with ASD-VSD. A pathway-oriented search revealed enrichment in the ciliome for both groups and a specific enrichment in Hedgehog and Jak-stat pathways among ASD+VSD patients. These genes or related pathways are therefore potentially involved in normal cardiogenesis as well as in cardiac malformations observed in individuals with trisomy 21. PMID:22912673

  16. Molecular signatures of cardiac defects in Down syndrome lymphoblastoid cell lines suggest altered ciliome and Hedgehog pathways.

    Directory of Open Access Journals (Sweden)

    Clémentine Ripoll

    Full Text Available Forty percent of people with Down syndrome exhibit heart defects, most often an atrioventricular septal defect (AVSD and less frequently a ventricular septal defect (VSD or atrial septal defect (ASD. Lymphoblastoid cell lines (LCLs were established from lymphocytes of individuals with trisomy 21, the chromosomal abnormality causing Down syndrome. Gene expression profiles generated from DNA microarrays of LCLs from individuals without heart defects (CHD(-; n = 22 were compared with those of LCLs from patients with cardiac malformations (CHD(+; n = 21. After quantile normalization, principal component analysis revealed that AVSD carriers could be distinguished from a combined group of ASD or VSD (ASD+VSD carriers. From 9,758 expressed genes, we identified 889 and 1,016 genes differentially expressed between CHD(- and AVSD and CHD(- and ASD+VSD, respectively, with only 119 genes in common. A specific chromosomal enrichment was found in each group of affected genes. Among the differentially expressed genes, more than 65% are expressed in human or mouse fetal heart tissues (GEO dataset. Additional LCLs from new groups of AVSD and ASD+VSD patients were analyzed by quantitative PCR; observed expression ratios were similar to microarray results. Analysis of GO categories revealed enrichment of genes from pathways regulating clathrin-mediated endocytosis in patients with AVSD and of genes involved in semaphorin-plexin-driven cardiogenesis and the formation of cytoplasmic microtubules in patients with ASD-VSD. A pathway-oriented search revealed enrichment in the ciliome for both groups and a specific enrichment in Hedgehog and Jak-stat pathways among ASD+VSD patients. These genes or related pathways are therefore potentially involved in normal cardiogenesis as well as in cardiac malformations observed in individuals with trisomy 21.

  17. Exon expression in lymphoblastoid cell lines from subjects with schizophrenia before and after glucose deprivation

    Directory of Open Access Journals (Sweden)

    Martin Maureen V

    2009-09-01

    Full Text Available Abstract Background The purpose of this study was to examine the effects of glucose reduction stress on lymphoblastic cell line (LCL gene expression in subjects with schizophrenia compared to non-psychotic relatives. Methods LCLs were grown under two glucose conditions to measure the effects of glucose reduction stress on exon expression in subjects with schizophrenia compared to unaffected family member controls. A second aim of this project was to identify cis-regulated transcripts associated with diagnosis. Results There were a total of 122 transcripts with significant diagnosis by probeset interaction effects and 328 transcripts with glucose deprivation by probeset interaction probeset effects after corrections for multiple comparisons. There were 8 transcripts with expression significantly affected by the interaction between diagnosis and glucose deprivation and probeset after correction for multiple comparisons. The overall validation rate by qPCR of 13 diagnosis effect genes identified through microarray was 62%, and all genes tested by qPCR showed concordant up- or down-regulation by qPCR and microarray. We assessed brain gene expression of five genes found to be altered by diagnosis and glucose deprivation in LCLs and found a significant decrease in expression of one gene, glutaminase, in the dorsolateral prefrontal cortex (DLPFC. One SNP with previously identified regulation by a 3' UTR SNP was found to influence IRF5 expression in both brain and lymphocytes. The relationship between the 3' UTR rs10954213 genotype and IRF5 expression was significant in LCLs (p = 0.0001, DLPFC (p = 0.007, and anterior cingulate cortex (p = 0.002. Conclusion Experimental manipulation of cells lines from subjects with schizophrenia may be a useful approach to explore stress related gene expression alterations in schizophrenia and to identify SNP variants associated with gene expression.

  18. Genome-wide transcriptional profiling reveals microRNA-correlated genes and biological processes in human lymphoblastoid cell lines.

    Directory of Open Access Journals (Sweden)

    Liang Wang

    Full Text Available BACKGROUND: Expression level of many genes shows abundant natural variation in human populations. The variations in gene expression are believed to contribute to phenotypic differences. Emerging evidence has shown that microRNAs (miRNAs are one of the key regulators of gene expression. However, past studies have focused on the miRNA target genes and used loss- or gain-of-function approach that may not reflect natural association between miRNA and mRNAs. METHODOLOGY/PRINCIPAL FINDINGS: To examine miRNA regulatory effect on global gene expression under endogenous condition, we performed pair-wise correlation coefficient analysis on expression levels of 366 miRNAs and 14,174 messenger RNAs (mRNAs in 90 immortalized lymphoblastoid cell lines, and observed significant correlations between the two species of RNA transcripts. We identified a total of 7,207 significantly correlated miRNA-mRNA pairs (false discovery rate q<0.01. Of those, 4,085 pairs showed positive correlations while 3,122 pairs showed negative correlations. Gene ontology analyses on the miRNA-correlated genes revealed significant enrichments in several biological processes related to cell cycle, cell communication and signal transduction. Individually, each of three miRNAs (miR-331, -98 and -33b demonstrated significant correlation with the genes in cell cycle-related biological processes, which is consistent with important role of miRNAs in cell cycle regulation. CONCLUSIONS/SIGNIFICANCE: This study demonstrates feasibility of using naturally expressed transcript profiles to identify endogenous correlation between miRNA and miRNA. By applying this genome-wide approach, we have identified thousands of miRNA-correlated genes and revealed potential role of miRNAs in several important cellular functions. The study results along with accompanying data sets will provide a wealth of high-throughput data to further evaluate the miRNA-regulated genes and eventually in phenotypic variations of

  19. Proliferation rates and gene expression profiles in human lymphoblastoid cell lines from patients with depression characterized in response to antidepressant drug therapy.

    Science.gov (United States)

    Breitfeld, J; Scholl, C; Steffens, M; Brandenburg, K; Probst-Schendzielorz, K; Efimkina, O; Gurwitz, D; Ising, M; Holsboer, F; Lucae, S; Stingl, J C

    2016-11-15

    The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin-a gene involved in neuronal stem cell regeneration-were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy.

  20. Proliferation rates and gene expression profiles in human lymphoblastoid cell lines from patients with depression characterized in response to antidepressant drug therapy

    Science.gov (United States)

    Breitfeld, J; Scholl, C; Steffens, M; Brandenburg, K; Probst-Schendzielorz, K; Efimkina, O; Gurwitz, D; Ising, M; Holsboer, F; Lucae, S; Stingl, J C

    2016-01-01

    The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin—a gene involved in neuronal stem cell regeneration—were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy. PMID:27845776

  1. Repair of DNA lesions induced by ultraviolet irradiation and aromatic amines in normal and repair-deficient human lymphoblastoid cell lines

    DEFF Research Database (Denmark)

    Stevnsner, Tinna; Frandsen, Henrik; Autrup, Herman

    1995-01-01

    A host cell reactivation (HCR) assay was employed to study the capacity of a normal and three repair-deficient human lymphoblastoid cell lines to repair DNA damage induced by UV irradiation and the aromatic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and N-acetyl-2-aminofluorene....... In the XP-D cell line, which had practically no DNA repair capacity, AAF adducts had a more potent inhibitory effect on gene expression than UV and PhIP adducts. When corrected for this inhibitory effect, the wild-type, XP-C and CS-B cell lines repaired low levels of AAF and UV adducts with similar...

  2. Morphologic, immunologic, enzymehistochemical and chromosomal analysis of a cell line derived from Hodgkin's disease : Evidence for a B-cell origin of Sternberg-Reed cells

    NARCIS (Netherlands)

    Poppema, Sibrand; de Jong, Bauke; Atmosoerodjo, Jane; Idenburg, Vera; Visser, Lydia; de Ley, Lou

    1985-01-01

    Cell lines derived from Hodgkin's disease may provide a clue to the nature of Sternberg-Reed cells. In the current study, the establishment of an Epstein-Barr-virus-negative lymphoblastoid cell line, derived from the pleural fluid of a patient with the nodular sclerosis type of Hodgkin's disease, is

  3. The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.

    Science.gov (United States)

    Ohashi, Makoto; Holthaus, Amy M; Calderwood, Michael A; Lai, Chiou-Yan; Krastins, Bryan; Sarracino, David; Johannsen, Eric

    2015-04-01

    The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16(INK4A) and p14(ARF) by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14(ARF) promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14(ARF) promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation.

  4. Induction of epstein-barr virus (EBV lytic cycle in vitro causes lipid peroxidation, protein oxidation and DNA damage in lymphoblastoid B cell lines

    Directory of Open Access Journals (Sweden)

    benmansour Riadh

    2011-07-01

    Full Text Available Abstract Background We investigated the oxidative modifications of lipids, proteins and DNA, potential molecular targets of oxidative stress, in two lymphoblastoid cell lines: B95-8 and Raji, after EBV lytic cycle induction. Conjugated dienes level was measured as biomarker of lipid peroxidation. Malondialdehyde adduct and protein carbonyl levels, as well as protein thiol levels were measured as biomarkers of protein oxidation. DNA fragmentation was evaluated as biomarker of DNA oxidation. Results After 48 h (peak of lytic cycle, a significant increase in conjugated dienes level was observed in B95-8 and Raji cell lines (p = 0.0001 and p = 0.019 respectively. Malondialdehyde adduct, protein carbonyl levels were increased in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls (MDA-adduct: p = 0.008 and p = 0.006 respectively; Carbonyl: p = 0.003 and p = 0.0039 respectively. Proteins thiol levels were decreased by induction in B95-8 and Raji cell lines (p = 0.046; p = 0.002 respectively. DNA fragmentation was also detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls. Conclusion The results of this study demonstrate the presence of increased combined oxidative modifications in lipids, proteins in B95-8 and Raji cells lines after EBV lytic cycle induction. These results suggest that lipid peroxidation, protein oxidation and DNA fragmentation are generally induced during EBV lytic cycle induction and probably contribute to the cytopathic effect of EBV.

  5. Gene expression profiling of lymphoblastoid cell lines from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes

    Directory of Open Access Journals (Sweden)

    Lee Norman H

    2006-05-01

    Full Text Available Abstract Background The autism spectrum encompasses a set of complex multigenic developmental disorders that severely impact the development of language, non-verbal communication, and social skills, and are associated with odd, stereotyped, repetitive behavior and restricted interests. To date, diagnosis of these neurologically based disorders relies predominantly upon behavioral observations often prompted by delayed speech or aberrant behavior, and there are no known genes that can serve as definitive biomarkers for the disorders. Results Here we demonstrate, for the first time, that lymphoblastoid cell lines from monozygotic twins discordant with respect to severity of autism and/or language impairment exhibit differential gene expression patterns on DNA microarrays. Furthermore, we show that genes important to the development, structure, and/or function of the nervous system are among the most differentially expressed genes, and that many of these genes map closely in silico to chromosomal regions containing previously reported autism candidate genes or quantitative trait loci. Conclusion Our results provide evidence that novel candidate genes for autism may be differentially expressed in lymphoid cell lines from individuals with autism spectrum disorders. This finding further suggests the possibility of developing a molecular screen for autism based on expressed biomarkers in peripheral blood lymphocytes, an easily accessible tissue. In addition, gene networks are identified that may play a role in the pathophysiology of autism.

  6. Host cell reactivation of uv- and X-ray-damaged herpes simplex virus by Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, E.E. (Temple Univ. School of Medicine, Philadelphia, PA); Long, W.K.

    1981-12-01

    The efficacy of using an infected centers assay, employing herpes simplex virus-infected, Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) as components, to study host cell reactivation has been explored. Herpes simplex virus type 1 (HSV-1) was shown through the infected centers assay to have detectable but varying ability to lytically infect LCLs established from chromosomal breakage syndromes or closely related genetic disorders. The rate of HSV inactivation by ultraviolet (uv) irradiation was faster in LCLs established from Cockaynes's syndrome than in normal LCLs, and faster still in LCLs established from xeroderma pigmentosum. These results indicate that Cockayne's syndrome, while having what appears to be quantitatively normal levels of uv-induced DNA repair replication, shows decreased ability to host cell reactivated uv-damaged HSV. In direct contrast, X-irradiated HSV showed identical survival when assayed on normal LCLs or LCLs established from ataxia telangiectasia showing increased sensitivity to X irradiation as measured by colony formation. Through the infected centers assay, it has also been possible to demonstrate low levels of multiplicity reactivation of mutagen-damaged HSV in permanently proliferating LCLs.

  7. Insulin-like Growth Factor 1 Differentially Affects Lithium Sensitivity of Lymphoblastoid Cell Lines from Lithium Responder and Non-responder Bipolar Disorder Patients.

    Science.gov (United States)

    Milanesi, Elena; Hadar, Adva; Maffioletti, Elisabetta; Werner, Haim; Shomron, Noam; Gennarelli, Massimo; Schulze, Thomas G; Costa, Marta; Del Zompo, Maria; Squassina, Alessio; Gurwitz, David

    2015-07-01

    Bipolar disorder (BD) is a chronic psychiatric illness with an unknown etiology. Lithium is considered the cornerstone in the management of BD, though about 50-60 % of patients do not respond sufficiently to chronic treatment. Insulin-like growth factor 1 (IGF1) has been identified as a candidate gene for BD susceptibility, and its low expression has been suggested as a putative biomarker for lithium unresponsiveness. In this study, we examined the in vitro effects of insulin-like growth factor 1 (IGF-1) on lithium sensitivity in lymphoblastoid cell lines (LCLs) from lithium responder (R) and non-responder (NR) bipolar patients. Moreover, we evaluated levels of microRNA let-7c, a small RNA predicted to target IGF1. We found that exogenous IGF-1 added to serum-free media increased lithium sensitivity selectively in LCLs from NR BD patients. However, no significant differences were observed when comparing let-7c expression in LCLs from R vs. NR BD patients. Our data support a key role for IGF-1 in lithium resistance/response in the treatment of bipolar disorder.

  8. Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain.

    Science.gov (United States)

    Nguyen, AnhThu; Rauch, Tibor A; Pfeifer, Gerd P; Hu, Valerie W

    2010-08-01

    Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.

  9. Instability of the expanded (CTG){sub n} repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Ashizawa, Tetsuo; Patel, B.J.; Monckton, D.G. [Baylor College of Medicine, Houston, TX (United States)] [and other

    1996-08-15

    The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG){sub n}, in the 3{prime}-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG). repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10{sup 6} cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG), repeat region. Two types of mutations of the expanded (CTG){sub n} repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG){sub n} repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG){sub n} repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the so matic heterogeneity of the (CTG), repeat size observe in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG). repeats in DM. 24 refs., 4 figs.

  10. Induction of p16(INK4a) is the major barrier to proliferation when Epstein-Barr virus (EBV) transforms primary B cells into lymphoblastoid cell lines.

    Science.gov (United States)

    Skalska, Lenka; White, Robert E; Parker, Gillian A; Turro, Ernest; Sinclair, Alison J; Paschos, Kostas; Allday, Martin J

    2013-02-01

    To explore the role of p16(INK4a) as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16(INK4a) and p14(ARF). Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16(INK4a) protein but expressing a functional 14(ARF)-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C--in cooperation with EBNA3A--despite the absence of functional p16(INK4a). Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16(INK4a) and growth arrest, EBNA3C inactivation in the p16(INK4a)-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16(INK4a)-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16(INK4a) expression and concomitant block to proliferation 2-4 weeks post-infection. If cells are p16(INK4a)-null, functional EBNA3C is dispensable for the outgrowth of LCLs.

  11. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress

    Science.gov (United States)

    Fenech, Michael F.

    2013-01-01

    Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case–sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally

  12. Induction of p16(INK4a is the major barrier to proliferation when Epstein-Barr virus (EBV transforms primary B cells into lymphoblastoid cell lines.

    Directory of Open Access Journals (Sweden)

    Lenka Skalska

    2013-02-01

    Full Text Available To explore the role of p16(INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16(INK4a and p14(ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT, we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs lacking active p16(INK4a protein but expressing a functional 14(ARF-fusion protein (p14/p16. The INK4a locus is epigenetically repressed by EBNA3C--in cooperation with EBNA3A--despite the absence of functional p16(INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16(INK4a and growth arrest, EBNA3C inactivation in the p16(INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16(INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16(INK4a expression and concomitant block to proliferation 2-4 weeks post-infection. If cells are p16(INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs.

  13. Unsuitability of lymphoblastoid cell lines as surrogate of cryopreserved isolated lymphocytes for the analysis of DNA double-strand break repair activity

    Energy Technology Data Exchange (ETDEWEB)

    Zijno, Andrea [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Porcedda, Paola [Department of Clinical and Biological Sciences, University of Turin (Italy); Saini, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Allione, Alessandra [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Garofalo, Bruno; Marcon, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Guarrera, Simonetta [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Turinetto, Valentina; Minieri, Valentina [Department of Clinical and Biological Sciences, University of Turin (Italy); Funaro, Ada [Department of Genetics, Biology and Biochemistry, University of Turin (Italy); Crebelli, Riccardo [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Giachino, Claudia [Department of Clinical and Biological Sciences, University of Turin (Italy); Matullo, Giuseppe, E-mail: giuseppe.matullo@unito.it [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Department of Genetics, Biology and Biochemistry, University of Turin (Italy)

    2010-02-03

    As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by {gamma}-rays and DNA repair capacity were evaluated in unstimulated (G{sub 0}) and mitogen-simulated (G{sub 2}) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G{sub 0}-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G{sub 2}-treated PBMC compared to LCL. Concerning {gamma}-H2AX measurements, phosphorylation levels 1 h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of {gamma}-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype

  14. Necrosis is increased in lymphoblastoid cell lines from children with autism compared with their non-autistic siblings under conditions of oxidative and nitrosative stress.

    Science.gov (United States)

    Main, Penelope A E; Thomas, Philip; Esterman, Adrian; Fenech, Michael F

    2013-07-01

    Autism spectrum disorders are a heterogeneous group of neurodevelopmental conditions characterised by impairments in reciprocal social interaction, communication and stereotyped behaviours. As increased DNA damage events have been observed in a range of other neurological disorders, it was hypothesised that they would be elevated in lymphoblastoid cell lines (LCLs) obtained from children with autism compared with their non-autistic siblings. Six case-sibling pairs of LCLs from children with autistic disorder and their non-autistic siblings were obtained from the Autism Genetic Resource Exchange (AGRE) and cultured in standard RPMI-1640 tissue culture medium. Cells were exposed to medium containing either 0, 25, 50, 100 and 200 µM hydrogen peroxide (an oxidative stressor) or 0, 5, 10, 20 and 40 µM s-nitroprusside (a nitric oxide producer) for 1h. Following exposure, the cells were microscopically scored for DNA damage, cytostasis and cytotoxicity biomarkers as measured using the cytokinesis-block micronucleus cytome assay. Necrosis was significantly increased in cases relative to controls when exposed to oxidative and nitrosative stress (P = 0.001 and 0.01, respectively). Nuclear division index was significantly lower in LCLs from children with autistic disorder than their non-autistic siblings when exposed to hydrogen peroxide (P = 0.016), but there was no difference in apoptosis, micronucleus frequency, nucleoplasmic bridges or nuclear buds. Exposure to s-nitroprusside significantly increased the number of micronuclei in non-autistic siblings compared with cases (P = 0.003); however, other DNA damage biomarkers, apoptosis and nuclear division did not differ significantly between groups. The findings of this study show (i) that LCLs from children with autism are more sensitive to necrosis under conditions of oxidative and nitrosative stress than their non-autistic siblings and (ii) refutes the hypothesis that children with autistic disorder are abnormally

  15. Epstein-Barr Viruses (EBVs) Deficient in EBV-Encoded RNAs Have Higher Levels of Latent Membrane Protein 2 RNA Expression in Lymphoblastoid Cell Lines and Efficiently Establish Persistent Infections in Humanized Mice.

    Science.gov (United States)

    Gregorovic, Goran; Boulden, Elizabeth A; Bosshard, Rachel; Elgueta Karstegl, Claudio; Skalsky, Rebecca; Cullen, Bryan R; Gujer, Cornelia; Rämer, Patrick; Münz, Christian; Farrell, Paul J

    2015-11-01

    Functions of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) were tested in lymphoblastoid cell lines containing EBER mutants of EBV. Binding of EBER1 to ribosomal protein L22 (RPL22) was confirmed. Deletion of EBER1 or EBER2 correlated with increased levels of cytoplasmic EBV LMP2 RNA and with small effects on specific cellular microRNA (miRNA) levels, but protein levels of LMP1 and LMP2A were not affected. Wild-type EBV and EBER deletion EBV had approximately equal abilities to infect immunodeficient mice reconstituted with a human hematopoietic system.

  16. BRCA1 and BRCA2 missense variants of high and low clinical significance influence lymphoblastoid cell line post-irradiation gene expression.

    Directory of Open Access Journals (Sweden)

    Nic Waddell

    2008-05-01

    Full Text Available The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases. 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS. BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82-94%, poor for BRCAX with an LCS (40-50%, and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71-100%. This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.

  17. Cadmium chloride, benzo[a]pyrene and cyclophosphamide tested in the in vitro mammalian cell micronucleus test (MNvit) in the human lymphoblastoid cell line TK6 at Covance laboratories, Harrogate UK in support of OECD draft Test Guideline 487.

    Science.gov (United States)

    Fowler, Paul; Whitwell, James; Jeffrey, Laura; Young, Jamie; Smith, Katie; Kirkland, David

    2010-10-29

    The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the human lymphoblastoid cell line TK6. Cadmium chloride (an inorganic carcinogen), benzo[a]pyrene (a polycyclic aromatic hydrocarbon requiring metabolic activation) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, capable of detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in the in vitro micronucleus assay.

  18. Mitomycin C, 5-fluoruracil, colchicine and etoposide tested in the in vitro mammalian cell micronucleus test (MNvit) in the human lymphoblastoid cell line TK6 at Novartis in support of OECD draft Test Guideline 487.

    Science.gov (United States)

    Elhajouji, Azeddine

    2010-10-29

    The following reference genotoxic agents were tested in the in vitro micronucleus test, at Novartis, Basel, Switzerland. Mitomycin C, 5-fluoruracil, colchicine and etoposide were tested in the human lymphoblastoid cell line TK6, with and without cytokinesis block (in the presence of cytochalasin B). This was done in support of the toxicity measures recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) and was part of an international collaborative work. As toxicity measures, detecting cytostasis and cell death, relative cell counts (RCC), relative increase in cell counts (RICC), and relative population doubling (RPD) were used for treatments in the absence of cytokinesis block, and replication index (RI) or cytokinesis-blocked proliferation in the presence of cytokinesis block. All four reference agents were positive in the assay with and without cytokinesis block at concentrations giving approximately 50% toxicity or less as assessed by all of the toxicity measures used. Accordingly, the results of this work support the use of relative population doubling and relative increase in cell counts, as well as relative cell counts, as appropriate measures of toxicity for the non-cytokinesis-blocked in vitro micronucleus assay.

  19. The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

    Science.gov (United States)

    Schwartz, Jeffrey L.; Jordan, Robert; Evans, Helen H.; Lenarczyk, Marek; Liber, Howard

    2003-01-01

    The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

  20. Deriving the Regression Line with Algebra

    Science.gov (United States)

    Quintanilla, John A.

    2017-01-01

    Exploration with spreadsheets and reliance on previous skills can lead students to determine the line of best fit. To perform linear regression on a set of data, students in Algebra 2 (or, in principle, Algebra 1) do not have to settle for using the mysterious "black box" of their graphing calculators (or other classroom technologies).…

  1. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    Science.gov (United States)

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  2. Reduction of 8-oxodGTP in the nucleotide pool by hMTH1 leads to reduction in mutations in the human lymphoblastoid cell line TK6 exposed to UVA

    Energy Technology Data Exchange (ETDEWEB)

    Fotouhi, Asal; Skioeld, Sara; Shakeri-Manesh, Sara; Osterman-Golkar, Siv; Wojcik, Andrzej; Jenssen, Dag; Harms-Ringdahl, Mats [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, SE-10691, Stockholm (Sweden); Haghdoost, Siamak, E-mail: siamak.haghdoost@gmt.su.se [Centre for Radiation Protection Research, Department of Genetics, Microbiology and Toxicology, Stockholm University, SE-10691, Stockholm (Sweden)

    2011-10-01

    UVA has been suggested to play an important role in UV-induced mutagenesis. The mechanisms by which UVA induces mutations are still a matter of debate. Our aim was to investigate the protective capacity of hMTH1, a nucleotide pool sanitization enzyme with 8-oxodGTPase activity. Human B lymphoblastoid cells were stably transfected with shRNA directed against hMTH1. Clonogenic survival, mutations, intracellular and extracellular levels of 8-oxodG (8-oxo-7, 8-dihydro-2'-deoxyguanosine) and dG in the nucleotide pool of UVA-irradiated transfected and non-transfected cells were investigated. Mutations were determined in the thymidine kinase locus. Intracellular 8-oxodG and dG were measured using a modified ELISA and HPLC, respectively, after extraction of the nucleotide pool and conversion of nucleotides to their corresponding nucleosides. 8-oxodG in the medium was measured using ELISA. UVA-induced mutations were significantly higher while the survival was slightly lower in transfected compared to non-transfected cells. The increased mutation rate in transfected cells at increased exposure correlated with enhanced levels of 8-oxodG in the nucleotide pool, and a somewhat reduced level of 8-oxodG in the medium. The results indicate that the nucleotide pool is a significant target for UVA-induced mutations and implicates that hMTH1 plays an important role in protecting cells from UVA-induced oxidative stress.

  3. A One Line Derivation of EGARCH

    Directory of Open Access Journals (Sweden)

    Michael McAleer

    2014-06-01

    Full Text Available One of the most popular univariate asymmetric conditional volatility models is the exponential GARCH (or EGARCH specification. In addition to asymmetry, which captures the different effects on conditional volatility of positive and negative effects of equal magnitude, EGARCH can also accommodate leverage, which is the negative correlation between returns shocks and subsequent shocks to volatility. However, the statistical properties of the (quasi- maximum likelihood estimator of the EGARCH parameters are not available under general conditions, but rather only for special cases under highly restrictive and unverifiable conditions. It is often argued heuristically that the reason for the lack of general statistical properties arises from the presence in the model of an absolute value of a function of the parameters, which does not permit analytical derivatives, and hence does not permit (quasi- maximum likelihood estimation. It is shown in this paper for the non-leverage case that: (1 the EGARCH model can be derived from a random coefficient complex nonlinear moving average (RCCNMA process; and (2 the reason for the lack of statistical properties of the estimators of EGARCH under general conditions is that the stationarity and invertibility conditions for the RCCNMA process are not known.

  4. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

    Science.gov (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans

    2016-01-01

    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

  5. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  6. Human NK cells activated by EBV+ lymphoblastoid cells overcome anti-apoptotic mechanisms of drug resistance in haematological cancer cells

    Science.gov (United States)

    Sánchez-Martínez, Diego; Azaceta, Gemma; Muntasell, Aura; Aguiló, Nacho; Núñez, David; Gálvez, Eva M; Naval, Javier; Anel, Alberto; Palomera, Luis; Vilches, Carlos; Marzo, Isabel; Villalba, Martín; Pardo, Julián

    2015-01-01

    Natural killer (NK) cells recognize and eliminate transformed or infected cells that have downregulated MHC class-I and express specific activating ligands. Recent evidence indicates that allogeneic NK cells are useful to eliminate haematological cancer cells independently of MHC-I expression. However, it is unclear if transformed cells expressing mutations that confer anti-apoptotic properties and chemoresistance will be susceptible to NK cells. Allogeneic primary human NK cells were activated using different protocols and prospectively tested for their ability to eliminate diverse mutant haematological and apoptotic-resistant cancer cell lines as well as patient-derived B-cell chronic lymphocytic leukemia cells with chemotherapy multiresistance. Here, we show that human NK cells from healthy donors activated in vitro with Epstein Barr virus positive (EBV+)-lymphoblastoid cells display an enhanced cytotoxic and proliferative potential in comparison to other protocols of activation such a K562 cells plus interleukin (IL)2. This enhancement enables them to kill more efficiently a variety of haematological cancer cell lines, including a panel of transfectants that mimic natural mutations leading to oncogenic transformation and chemoresistance (e.g., overexpression of Bcl-2, Bcl-XL and Mcl-1 or downregulation of p53, Bak/Bax or caspase activity). The effect was also observed against blasts from B-cell chronic lymphocytic leukemia patients showing multi-resistance to chemotherapy. Our findings demonstrate that particular in vitro activated NK cells may overcome anti-apoptotic mechanisms and oncogenic alterations frequently occurring in transformed cells, pointing toward the use of EBV+-lymphoblastoid cells as a desirable strategy to activate NK cells in vitro for the purpose of treating haematological neoplasia with poor prognosis. PMID:25949911

  7. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    Science.gov (United States)

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  8. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1–60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  9. The Derivation of Skeleton Lines for Terrain Features

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The geometric and physical analysis methods are conventional methods for the derivation of skeleton lines in the fields of cartography,digital photogrammetry,and related areas.This paper proposes a stepwise approach that uses the physical analysis method in the first stage and the geometric analysis method in the subsequent stage.The physical analysis method analyses the terrain globally to obtain a rough set of skeleton lines for a terrain surface.The rough skeleton lines help to structure the ordering of feature points by the geometric analysis method.

  10. Optimized protocol for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

    2012-09-01

    For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure.

  11. Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s.

    Science.gov (United States)

    Styles, J A; Davies, A; Lim, C K; De Matteis, F; Stanley, L A; White, I N; Yuan, Z X; Smith, L L

    1994-01-01

    The clastogenicity of tamoxifen and toremifene was tested in six human lymphoblastoid cell lines each expressing increased monooxygenase activity associated with a specific transfected human cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 or CYP3A4). The chemicals were also tested in a cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, and in a cell line containing only the viral vector (Ho1). Dose-related increases in micronuclei were observed when cells expressing 2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. The positive responses in the cell lines were in the order MCL-5 > 2E1 > 3A4 > 2D6. Toremifene also gave positive results with 2E1, 3A4 and MCL-5 cells, although the responses were less marked and the positive effects required higher doses than with tamoxifen. A synthesized epoxide of tamoxifen was also tested in these cell lines and produced similar increases in the incidences of micronucleated cells. The increases in the responses observed with the epoxide were greater than with tamoxifen or toremifene. The P450 isoenzyme activities in these cells were in a range similar to those of human tumour-derived cell lines. Microsomes (1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells all metabolized tamoxifen. The major metabolite detected by HPLC was N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detected in cells with cytochrome P450 2E1 and 2D6. These results are consistent with the following conclusions. (1) Tamoxifen requires metabolic activation to DNA-reactive species by specific CYP monooxygenases in order to exert its genotoxic effects. (2) The positive clastogenic effects elicited in lymphoblastoid cells by tamoxifen epoxide suggest that the genotoxic (and possibly the carcinogenic) effects of tamoxifen may be due to one or more epoxide metabolites that are generated intracellularly, probably in close proximity to the nucleus. (3) Tamoxifen is

  12. Comparison of seven cell lines derived from human gastric carcinomas.

    Science.gov (United States)

    Motoyama, T; Hojo, H; Watanabe, H

    1986-01-01

    In an attempt to elucidate various histological features of gastric cancers, seven human gastric adenocarcinomas were studied in vitro and in nude mice. Growth pattern of each cultured cell line in vitro corresponded well to the histological type of parent tumor. The cell lines, MKN7, MKN74, and MKN28 derived from differentiated carcinomas showed morphological characteristics of intestinal differentiation in cell polarity and microvilli with core-filaments in vitro as well as in nude mice. However, they gradually diminished the characteristics in course of time. The cell lines, MKN 45 and OKAJIMA, derived from undifferentiated carcinomas, had natures of not only ordinary gastric mucosa but also intestinal metaplastic mucosa. They seem to have multipotentiality for differentiation, and preserved well the natures for long periods of culture. The KWS-I cell line composed of undifferentiated cells in vitro displayed the potential for differentiation in nude mice. However, the differentiation of KATO-III cells derived from a signet-ring cell carcinoma was suppressed in nude mice. The common abnormality of chromosome was not found, and the growth rate in vitro was not dependent on the histological type of parent tumor.

  13. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination.

  14. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  15. Formalizing Traceability and Derivability in Software Product Lines

    CERN Document Server

    Krishna, Shankara Narayanan; S., Ramesh; Mohalik, Swarup; Millo, Jean-Vivien

    2012-01-01

    In the literature, the definition of product in a Software Product Line (SPL) is based upon the notion of consistency of the constraints, imposed by variability and traceability relations on the elements of the SPL. In this paper, we contend that consistency does not model the natural semantics of the implementability relation between problem and solution spaces correctly. Therefore, we define when a feature can be {\\em derived} from a set of components . Using this, we define a product of the SPL by a pair, where all the features in the specification are derived from the components in the architecture. This notion of derivability is formulated in a simple yet expressive, abstract model of a productline with traceability relation. We then define a set of SPL analysis problems and show that these problems can be encoded as Quantified Boolean Formulas. Then, QSAT solvers like QUBE can be used to solve the analysis problems. We illustrate the methodology on a small fragment of a realistic productline.

  16. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  17. Targeting Epstein-Barr virus–transformed B lymphoblastoid cells using antibodies with T-cell receptor–like specificities

    Science.gov (United States)

    Lai, Junyun; Tan, Wei Jian; Too, Chien Tei; Choo, Joanna Ai Ling; Wong, Lan Hiong; Mustafa, Fatimah Bte; Srinivasan, Nalini; Lim, Angeline Pei Chiew; Zhong, Youjia; Gascoigne, Nicholas R. J.; Hanson, Brendon J.; Chan, Soh Ha; Chen, Jianzhu

    2016-01-01

    Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor–like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg−/− mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases. PMID:27338099

  18. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  19. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

  20. Physical conditions derived from OII recombination lines in planetary nebulae and their implications

    CERN Document Server

    Peimbert, Antonio; Delgado-Inglada, Gloria; Garcia-Rojas, Jorge; Peña, Miriam

    2014-01-01

    Based on high quality observations of multiplet V1 of OII and the NLTE atomic computations for OII we study the density and temperature of a sample of PNe. We find that, in general, the densities derived from recombination lines of OII are similar than the densities derived from forbidden lines. This implies that the signature for oxygen rich clumps of high density and low temperature is absent in most of the objects of our sample. Electron pressures derived from the hotter zones are similar or slightly larger than those derived from the colder zones, suggesting the presence of shock waves. The average temperatures and t2 values derived from H, He and O lines are similar and consistent with chemical homogeneity. These results suggest that the abundances of these objects are the ones derived from recombination lines.

  1. Population differences in the rate of proliferation of international HapMap cell lines.

    Science.gov (United States)

    Stark, Amy L; Zhang, Wei; Zhou, Tong; O'Donnell, Peter H; Beiswanger, Christine M; Huang, R Stephanie; Cox, Nancy J; Dolan, M Eileen

    2010-12-10

    The International HapMap Project is a resource for researchers containing genotype, sequencing, and expression information for EBV-transformed lymphoblastoid cell lines derived from populations across the world. The expansion of the HapMap beyond the four initial populations of Phase 2, referred to as Phase 3, has increased the sample number and ethnic diversity available for investigation. However, differences in the rate of cellular proliferation between the populations can serve as confounders in phenotype-genotype studies using these cell lines. Within the Phase 2 populations, the JPT and CHB cell lines grow faster (p HapMap panels into discovery and replication sets must take this into consideration.

  2. Rigorous theoretical derivation of lumped models to transmission line systems

    Institute of Scientific and Technical Information of China (English)

    Zhao Jixiang

    2012-01-01

    By virtue of the negative electric parameter concept,i.e.negative lumped resistance,inductance,conductance and capacitance (N-RLGC),the lumped equivalent models of transmission line systems,including the circuit model,two-port π-network and T-network,are given.We start from the N-segment-ladder-like equivalent networks composed distributed parameters,and achieve the input impedance in the form of a continued fraction.Utilizing the continued fraction theory,the expressions of input impedance are obtained under three kinds of extreme cases,i.e.the load impedances are equal to zero,infinity and characteristic impedance,respectively.When the number of segment N is limited to infinity,they are transformed to lumped elements.Comparison between the distributed model and lumped model of transmission lines,the expression of tanh yd,which is the key term in the transmission line equations,are obtained by RLGC,furthermore,according to input admittance,admittance matrix and ABCD matrix of transmission lines,the lumped equivalent circuit models,π-networks and T-networks have been given.The models are verified in the frequency and time domain,respectively,showing that the models are accurate and efficient.

  3. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Jie Du; Xiaoqing Gao; Li Deng; Nengbin Chang; Huailin Xiong; Yu Zheng

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro-tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su-pernatant were signiifcantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes-enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen-chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

  4. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  5. Centrosomal and mitotic abnormalities in cell lines derived from papillary thyroid cancer harboring specific gene alterations

    National Research Council Canada - National Science Library

    Maric, Irena; Viaggi, Silvia; Caria, Paola; Frau, Daniela V; Degan, Paolo; Vanni, Roberta

    2011-01-01

    .... We investigated the centrosome status and mitotic abnormalities in three thyroid carcinoma-derived cell lines, each maintaining the specific, biologically relevant gene alteration harbored by the parental tumors...

  6. Stellwagen Bank bathymetry - Percent slope derived from 5-meter bathymetric contour lines

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Percent slope of Stellwagen Bank bathymetry. Raster derived from 5-meter bathymetric contour lines (Quads 1-18). Collected on surveys carried out in 4 cruises 1994 -...

  7. Stellwagen Bank bathymetry - Degree slope derived from 5-meter bathymetric contour lines

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Degree slope of Stellwagen Bank bathymetry. Raster derived from 5-meter bathymetric contour lines (Quads 1-18). Collected on surveys carried out in 4 cruises 1994 -...

  8. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    Directory of Open Access Journals (Sweden)

    Létourneau Isabelle J

    2012-08-01

    Full Text Available Abstract Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133 were derived from solid tumor (TOV and ascites (OV, at specific time points at diagnosis and relapse (R. Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133, or cisplatin/topotecan (2295. Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2, the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R and OV2295(R2 cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian

  9. USDA 846-1 fractal melon and derived recombinant inbred lines

    Science.gov (United States)

    The Agricultural Research Service, United States Department of Agriculture announces the release of a melon (Cucumis melo L.) breeding line with highly branched, fractal-type architectural growth habit and 81 derived recombinant inbred lines (RIL). The indeterminate, monoecious USDA 846-1 produces 2...

  10. The derivation of on-line algorithms, with an application to finding palindromes

    NARCIS (Netherlands)

    Jeuring, J.T.

    1994-01-01

    A theory for the derivation of on-line algorithms is presented. The algorithms are derived in the Bird-Meertens calculus for program transformations. This calculus provides a concise functional notation for algorithms, and a few powerful theorems for proving equalities of functions. The

  11. Derivation of Soil Line Influence on Two-Band Vegetation Indices and Vegetation Isolines

    Directory of Open Access Journals (Sweden)

    Hiroki Yoshioka

    2009-11-01

    Full Text Available This paper introduces derivations of soil line influences on two-band vegetation indices (VIs and vegetation isolines in the red and near infra-red reflectance space. Soil line variations are described as changes in the soil line parameters (slope and offset and the red reflectance of the soil surface. A general form of a VI model equation written as a ratio of two linear functions (e.g., NDVI and SAVI was assumed. It was found that relative VI variations can be approximated by a linear combination of the three soil parameters. The derived expressions imply the possibility of estimating and correcting for soil-induced bias errors in VIs and their derived biophysical parameters, caused by the assumption of a general soil line, through the use of external data sources such as regional soil maps.

  12. No relationship between embryo morphology and successful derivation of human embryonic stem cell lines.

    Directory of Open Access Journals (Sweden)

    Susanne Ström

    Full Text Available BACKGROUND: The large number (30 of permanent human embryonic stem cell (hESC lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002-2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. METHODS: We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. RESULTS: Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. CONCLUSION: Even very poor quality embryos with few cells in the ICM can give origin to hESC lines.

  13. Derivation of the King's College London human embryonic stem cell lines.

    Science.gov (United States)

    Stephenson, Emma L; Braude, Peter R

    2010-04-01

    Since the derivation of the first human embryonic stem cell (hESC) line in 1998, there has been substantial interest in the potential of these cells for regenerative medicine and cell therapy and in the use of hESCs carrying clinically relevant genetic mutations as models for disease research and therapeutic target identification. There is still a need to improve derivation efficiency and further the understanding of the basic biology of these cells and to develop clinical grade culture systems with the aim of producing cell lines suitable for subsequent manipulation for therapy. The derivation of initial hESC lines at King's College London is discussed here, with focus on derivation methodology. Each of the derivations was distinctive. Although the stage and morphology of each blastocyst were generally similar in each attempt, the behaviour of the colonies was unpredictable; colony morphology and development was different with each attempt. Days 5, 6 and 7 blastocysts were used successfully, and the number of days until appearance of stem-like cells varied from 4 to 14 d. Routine characterisation analyses were performed on three lines, all of which displayed appropriate marker expression and survived cryopreservation-thaw cycles. From the lines discussed, four are at various stages of the deposition process with the UKSCB, one is pending submission and two are unsuitable for banking. Continued open and transparent reporting of results and collaborations will maximise the efficiency of derivation and facilitate the development of standardised protocols for the derivation and early culture of hESC lines.

  14. Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

    Science.gov (United States)

    Gardell, Alison M; Qin, Qin; Rice, Robert H; Li, Johnathan; Kültz, Dietmar

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

  15. Transcription of LINE-derived sequences in exercise-induced stress in horses.

    Science.gov (United States)

    Capomaccio, S; Verini-Supplizi, A; Galla, G; Vitulo, N; Barcaccia, G; Felicetti, M; Silvestrelli, M; Cappelli, K

    2010-12-01

    A large proportion of mammalian genomes is represented by transposable elements (TE), most of them being long interspersed nuclear elements 1 (LINE-1 or L1). An increased expression of LINE-1 elements may play an important role in cellular stress-related conditions exerting drastic effects on the mammalian transcriptome. To understand the impact of TE on the known horse transcriptome, we masked the horse EST database, pointing out that the amount is consistent with other major vertebrates. A previously developed transcript-derived fragments (TDFs) dataset, deriving from exercise-stimulated horse peripheral blood mononuclear cells (PBMCs), was found to be enriched with L1 (26.8% in terms of bp). We investigated the involvement of TDFs in exercise-induced stress through bioinformatics and gene expression analysis. Results indicate that LINE-derived sequences are not only highly but also differentially expressed during physical effort, hinting at interesting scenarios in the regulation of gene expression in relation to exercise.

  16. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  17. Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines.

    Science.gov (United States)

    Michishita, Masaki; Akiyoshi, Rui; Yoshimura, Hisashi; Katsumoto, Takuo; Ichikawa, Hitoshi; Ohkusu-Tsukada, Kozo; Nakagawa, Takayuki; Sasaki, Nobuo; Takahashi, Kimimasa

    2011-10-01

    There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44+CD24- population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10(4)sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.

  18. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

    DEFF Research Database (Denmark)

    Efrat, S; Linde, S; Kofod, Hans

    1988-01-01

    Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The ...... both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype....

  19. Derivation of the human embryonic stem cell line RCe008-A (RC-4

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe008-A (RC-4 was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is available.

  20. Derivation of the human embryonic stem cell line RCe012-A (RC-8)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Collins, D. M.; Greenshields, A.; H. Bradburn; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyoty...

  1. Derivation of the human embryonic stem cell line RCe006-A (RC-2)

    OpenAIRE

    P.A. De Sousa; B. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe006-A (RC-2) was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12). Mic...

  2. Derivation of the human embryonic stem cell line RCe011-A (RC-7)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Collins, D. M.; Bruce, K.; P. Dand; RUSSELL, G.; H. Bradburn; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe011-A (RC-7) was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group t...

  3. Derivation of the human embryonic stem cell line RCe007-A (RC-3

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe007-A (RC-3 was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available.

  4. Derivation of the human embryonic stem cell line RCe007-A (RC-3)

    OpenAIRE

    P.A. De Sousa; B. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe007-A (RC-3) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available.

  5. Derivation of the human embryonic stem cell line RCe010-A (RC-6)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Collins, D. M.; H. Bradburn; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe010-A (RC-6) was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group...

  6. Derivation of the human embryonic stem cell line RCe014-A (RC-10)

    OpenAIRE

    P.A. De Sousa; B.J. Tye; Bruce, K.; P. Dand; RUSSELL, G.; Collins, D. M.; Greenshields, A.; H. Bradburn; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group ty...

  7. Derivation of the human embryonic stem cell line RCe008-A (RC-4)

    OpenAIRE

    P.A. De Sousa; B. Tye; Bruce, K.; P. Dand; Gardner, J.; J.M. Downie; M. Bateman; A. Courtney

    2016-01-01

    The human embryonic stem cell line RCe008-A (RC-4) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is av...

  8. Derivation of the clinical grade human embryonic stem cell line RCe013-A (RC-9

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe013-A (RC-9 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following informed consent. RCe013-A (RC-9 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  9. Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe016-A (RC-12 was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12 shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15, which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

  10. Derivation of the clinical grade human embryonic stem cell line RCe019-A (RC-15

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe019-A (RC-15 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  11. Derivation of the clinical grade human embryonic stem cell line RCe015-A (RC-11

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe015-A (RC-11 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  12. Derivation of the clinical grade human embryonic stem cell line RCe021-A (RC-17

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-07-01

    Full Text Available The human embryonic stem cell line RCe021-A (RC-17 was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17 shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  13. Gene expression analysis in lymphoblasts derived from patients with autism spectrum disorder

    Directory of Open Access Journals (Sweden)

    Yasuda Yuka

    2011-05-01

    Full Text Available Abstract Background The autism spectrum disorders (ASDs are complex neurodevelopmental disorders that result in severe and pervasive impairment in the development of reciprocal social interaction and verbal and nonverbal communication skills. In addition, individuals with ASD have stereotypical behavior, interests and activities. Rare mutations of some genes, such as neuroligin (NLGN 3/4, neurexin (NRXN 1, SHANK3, MeCP2 and NHE9, have been reported to be associated with ASD. In the present study, we investigated whether alterations in mRNA expression levels of these genes could be found in lymphoblastoid cell lines derived from patients with ASD. Methods We measured mRNA expression levels of NLGN3/4, NRXN1, SHANK3, MeCP2, NHE9 and AKT1 in lymphoblastoid cells from 35 patients with ASD and 35 healthy controls, as well as from 45 patients with schizophrenia and 45 healthy controls, using real-time quantitative reverse transcriptase polymerase chain reaction assays. Results The mRNA expression levels of NLGN3 and SHANK3 normalized by β-actin or TBP were significantly decreased in the individuals with ASD compared to controls, whereas no difference was found in the mRNA expression level of MeCP2, NHE9 or AKT1. However, normalized NLGN3 and SHANK3 gene expression levels were not altered in patients with schizophrenia, and expression levels of NLGN4 and NRXN1 mRNA were not quantitatively measurable in lymphoblastoid cells. Conclusions Our results provide evidence that the NLGN3 and SHANK3 genes may be differentially expressed in lymphoblastoid cell lines from individuals with ASD compared to those from controls. These findings suggest the possibility that decreased mRNA expression levels of these genes might be involved in the pathophysiology of ASD in a substantial population of ASD patients.

  14. Generation and characterization of two iPSC lines from human epicardium-derived cells

    Directory of Open Access Journals (Sweden)

    Christina Paulitschek

    2017-04-01

    Full Text Available Human epicardium-derived cells (EPDC were reprogrammed to generate two iPSC lines, MCDU1i-EPDC and MCDU2i-EPDC, by nucleofection of episomal-based plasmids expressing the reprogramming factors OCT4, SOX2, KLF4, c-MYC, NANOG and LIN28. Pluripotency was confirmed in vitro by immunofluorescence analysis and embryoid body formation. The iPSC lines and the human embryonic stem cell line H1 show a Pearson correlation co-efficient of 0.951 (MCDU1i-EPDC and 0.937 (MCDU2i-EPDC as assessed by comparative transcriptome profiling.

  15. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  16. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1–60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  17. Derivation of Trisomy 21 affected human embryonic stem cell line Genea053

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea053 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analysis demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology and expressed pluripotent cell markers including 83% Nanog positive, 87% Oct4, 88% Tra1-60 and 98% SSEA4. The cell line was negative for Mycoplasma and visible contamination.

  18. Analysis of G-banding in tumor cell lines derived from human neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Junhua Zou; Yanhui Li

    2006-01-01

    BACKGROUND: The application of neural stem cell (NSC) is restricted because of its tumorigenesis, and the possible pathogenesis needs investigation.OBJECTIVE: To compare the differences of chromosomal G-banding between human NSCs (hNSCs) derived tumor cell line and hNSCs derived normal cell lines.DESIGN: A randomized controlled observation.SETTING: Building of Anatomy, Peking University Health Science Center.MATERIALS: The hNSC lines and hNSC-derived tumor cell lines were provided by the Research Center of Stem Cells, Peking University; DMEM/F12 (1:1) medium, N2 additive, B27 additive epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were produced by GIBCO BRL Company (USA); fetal bovine serum by HYCLONE Company (USA).METHODS: The experiments were carried out in the Department of Genetics, Peking University Health Science Center from February 2003 to July 2004. Human fetal striatal NSCs were inoculated hypodermically on the right scapular of nude mice; Normal human fetal striatal NSCs were cultured to 5-8 passages as controls. Karyotyping was performed on the 5th passage of hNSC-derived tumor cells at 6 weeks after hN-SC transplantation into nude mice (T1) and tumor cells at 15 weeks after transplantation (T2). Metaphase chromosomes were examined with microscope, G-banding cytogenetic analysis and karyotyping were performed according to the Cytoscan Karyotyping FISH and CGH software system (United biotechnology USA Corporation).MAIN OUTCOME MEASURES: G-banded analytical results of human fetal striatal nerve stem cells derived tumor cell lines (T1 and T2) of metaphase chromosomes were observed.RESULTS: ① Chromosome analysis of hNSC-derived tumor cell lines 1 (T1): Twenty-five well-spread metaphases were randomly selected for analysis. The karyotypes were 64, XX (8, 32%); 65, XX (1, 4%); 67,XX (5, 20%); 68, XX (11, 44%). The modal number of chromosomes in this cell lines was 68, which were all hypotriploid. The analysis of 8 G

  19. Electrosensory ampullary organs are lateral line placode-derived in bony fishes

    Science.gov (United States)

    Modrell, Melinda S.; Bemis, William E.; Northcutt, R. Glenn; Davis, Marcus C.; Baker, Clare V. H.

    2014-01-01

    Electroreception is an ancient subdivision of the lateral line sensory system, found in all major vertebrate groups (though lost in frogs, amniotes, and most ray-finned fishes). Electroreception is mediated by “hair cells” in ampullary organs, distributed in fields flanking lines of mechanosensory hair cell-containing neuromasts that detect local water movement. Neuromasts, and afferent neurons for both neuromasts and ampullary organs, develop from lateral line placodes. Although ampullary organs in the axolotl (a representative of the lobe-finned clade of bony fishes) are lateral line placode-derived, non-placodal origins have been proposed for electroreceptors in other taxa. Here we show morphological and molecular data describing lateral line system development in the basal ray-finned fish Polyodon spathula, and present fate-mapping data that conclusively demonstrate a lateral line placode origin for ampullary organs and neuromasts. Together with the axolotl data, this confirms that ampullary organs are ancestrally lateral line placode-derived in bony fishes. PMID:21988912

  20. Electrosensory ampullary organs are derived from lateral line placodes in bony fishes.

    Science.gov (United States)

    Modrell, Melinda S; Bemis, William E; Northcutt, R Glenn; Davis, Marcus C; Baker, Clare V H

    2011-10-11

    Electroreception is an ancient subdivision of the lateral line sensory system, found in all major vertebrate groups (though lost in frogs, amniotes and most ray-finned fishes). Electroreception is mediated by 'hair cells' in ampullary organs, distributed in fields flanking lines of mechanosensory hair cell-containing neuromasts that detect local water movement. Neuromasts, and afferent neurons for both neuromasts and ampullary organs, develop from lateral line placodes. Although ampullary organs in the axolotl (a representative of the lobe-finned clade of bony fishes) are lateral line placode-derived, non-placodal origins have been proposed for electroreceptors in other taxa. Here we show morphological and molecular data describing lateral line system development in the basal ray-finned fish Polyodon spathula, and present fate-mapping data that conclusively demonstrate a lateral line placode origin for ampullary organs and neuromasts. Together with the axolotl data, this confirms that ampullary organs are ancestrally lateral line placode-derived in bony fishes.

  1. The role of mitochondria in the radiation-induced bystander effect in human lymphoblastoid cells.

    Science.gov (United States)

    Rajendran, Sountharia; Harrison, Scott H; Thomas, Robert A; Tucker, James D

    2011-02-01

    Cells without intact mitochondrial DNA have been shown to lack the bystander effect, which is an energy-dependent process. We hypothesized that cells harboring mutations in mitochondrial genes responsible for ATP synthesis would show a decreased bystander effect compared to normal cells. Radiation-induced bystander effects were analyzed in two normal and four mitochondrial mutant human lymphoblastoid cells. Medium from previously irradiated cells (conditioned medium) was transferred to unirradiated cells from the respective cell lines and evaluated for the bystander effect using the cytokinesis-block micronucleus assay. Unlike normal cells that were used as a control, mitochondrial mutant cells neither generated nor responded to the bystander signals. The bystander effect was inhibited in normal cells by adding the mitochondrial inhibitors rotenone and oligomycin to the culture medium. Time-controlled blocking of the bystander effect by inhibitors was found to occur either for prolonged exposure to the inhibitor prior to irradiation with an immediate and subsequent removal of the inhibitors or immediate post-application of the inhibitor. Adding the inhibitors just prior to irradiation and removing them immediately after irradiation was uneventful. Fully functional mitochondrial metabolic capability may therefore be essential for the bystander effect.

  2. In vivo assay for the developmental competence of embryo-derived zebrafish cell lines

    NARCIS (Netherlands)

    Speksnijder, JE; Hage, WJ; Lanser, PH; Collodi, P; Zivkovic, D

    1997-01-01

    We have produced chimeric zebrafish embryos by transplanting permanent embryo-derived cell lines into blastula-stage embryos. Furthermore, we have established a fluorescent in vivo assay to monitor the developmental effects and fate of such transplanted cells using confocal laser scanning microscopy

  3. Use of RNAi silencing to target preconditioned glial cell line-derived neurotrophic factor in neuronal apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hongliang Guo; Zhongxin Xu; Xinhua Li; Jing Mang; Ying Xing; Jinting He; Guihua Xu; Shijun Yan; Lifeng Liu; Chunli Mei

    2011-01-01

    Several studies have suggested that exogenous glial cell line-derived neurotrophic factor may protect neurons from cerebral ischemic injury. However, the mechanisms underlying the neuroprotective effects of endogenous glial cell line-derived neurotrophic factor remain unclear. The present experiments sought to elucidate the influence of various conditioned media on neuronal apoptosis, using a normal culture medium for astrocytes, an astrocyte medium highly expressing glial cell line-derived neurotrophic factor, and an astrocyte medium in which glial cell line-derived neurotrophic factor expression was silenced using RNAi technology. The results confirmed that the use of RNAi silencing to target pretreated glial cell line-derived neurotrophic factor expression promoted neuronal apoptosis. In addition, oxygen and glucose deprivation preconditioning was found to upregulate glial cell line-derived neurotrophic factor expression, and significantly reduce neuronal apoptosis.

  4. Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

    DEFF Research Database (Denmark)

    Corrêa, Natássia C R; Kuasne, Hellen; Faria, Jerusa A Q A

    2013-01-01

    Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1...... and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted....... This shift in expression may be due to the selection of an 'establishment' phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines...

  5. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

    Directory of Open Access Journals (Sweden)

    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

  6. Characterization of human PGD blastocysts with unbalanced chromosomal translocations and human embryonic stem cell line derivation?

    Science.gov (United States)

    Frydman, N; Féraud, O; Bas, C; Amit, M; Frydman, R; Bennaceur-Griscelli, A; Tachdjian, G

    2009-01-01

    Novel embryonic stem cell lines derived from embryos carrying structural chromosomal abnormalities obtained after preimplantation genetic diagnosis (PGD) are of interest to study in terms of the influence of abnormalities on further development. A total of 22 unbalanced blastocysts obtained after PGD were analysed for structural chromosomal defects. Morphological description and chromosomal status of these blastocysts was established and they were used to derive human embryonic stem cell (ESC) lines. An outgrowth of cells was observed for six blastocysts (6/22; 27%). For two blastocysts, the exact morphology was unknown since they were at early stage, and for four blastocysts, the inner cell mass was clearly visible. Fifteen blastocysts carried an unbalanced chromosomal defect linked to a reciprocal translocation, resulting in a positive outgrowth of cells for five blastocysts. One human ESC line was obtained from a blastocyst carrying a partial chromosome-21 monosomy and a partial chromosome-1 trisomy. Six blastocysts carried an unbalanced chromosomal defect linked to a Robertsonian translocation, and one showed a positive outgrowth of cells. One blastocyst carried an unbalanced chromosomal defect linked to an insertion and no outgrowth was observed. The efficiency of deriving human ESC lines with constitutional chromosomal disorders was low and probably depends on the initial morphological aspect of the blastocysts and/or the type of the chromosomal disorders.

  7. Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts

    Institute of Scientific and Technical Information of China (English)

    Chunliang Li; Ying Yang; Junjie Gu; Yu Ma; Ying Jin

    2009-01-01

    Embryonic stem (ES) cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy. ES cells of mouse and human have been successfully generated and applied in a wide range of research. However, no genuine ES cell lines have been obtained from rat to date. In this study, we identified pluripotent cells in early rat embryos using specific antibodies against markers of pluripotent stem cells. Subsequently, by modifying the culture medium for rat blastocysts, we derived pluripotent rat ES-llke cell lines, which expressed pluripotency markers and formed embryoid bodies (EBs) in vitro. Importantly, these rat ES-like cells were able to produce teratomas. Both EBs and teratomas contained tissues from all three embryonic germ layers, in addition, from the rat ES-like cells, we derived a rat primitive endoderm (PrE) cell line. Furthermore, we conducted transcriptional profiling of the rat ES-like cells and identified the unique molecular signature of the rat pluripotent stem cells. Our analysis demonstrates that multiple signaling pathways, including the BMP, Activin and roTOR pathways, may be involved in keeping the rat ES-like cells in an undifferentiated state. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying pluripotency and guide us in the appropriate manipulation of ES cells from a particular species.

  8. Morphological and molecular characterization of an undifferentiated soft tissue sarcoma cell line and derivative clones.

    Science.gov (United States)

    Taubert, H; Schmidt, H; Wurl, P; Hinze, R; Meye, A; Bache, M; Berger, D; Holzhausen, H; Dralle, H; Rath, F

    1997-09-01

    From an undifferentiated soft tissue sarcoma (STS) a cell line designated US8-93 has been established. At subcloning the cell line US8-93 three different lines (US8-93A, B and C) could be set up. In a subsequent study characteristics for ultrastructure, growth, cell cycle distribution, karyotype, protein overexpression detected by immunohistochemistry (IHC) and p53 mutational status were determined. The cell line US8-93 as well as subclones contain mainly bipolar spindle-shaped cells and additionally some polygonal and multinucleated cells. Cells possess the characteristics of primitive mesenchymal cells based on their positive reactions with anti-vimentin and negative reactions for desmin, cytokeratin, myoglobin, S100, and NSE, implying a classification as an undifferentiated STS. Cytogenetic analysis revealed nearly diploid cells with several structural and numerical aberrations for chromosomes 1, 3, 4, 6, 9, 10, 12, 13, 15 and 18. IHC positivity was found for the tumor suppressor proteins p53 and Rb, the oncogene products Bcl-2, K-ras, N-ras, P-glycoprotein Mdr-1 and MDM-2. In the p53 gene a nonsense mutation in exon 4 was detected, that was confirmed in the original primary tumor and in three derivative clonal lines. The described STS cell line represents a valuable supplementation to the relatively small number of human STS cell lines currently available and may also provide a good in vitro model for studies of STS tumorigenesis in respect to a mutated p53 gene.

  9. Hypoxia-regulated gene expression explains differences between melanoma cell line-derived xenografts and patient-derived xenografts.

    Science.gov (United States)

    Bhadury, Joydeep; Einarsdottir, Berglind O; Podraza, Agnieszka; Bagge, Roger Olofsson; Stierner, Ulrika; Ny, Lars; Dávila López, Marcela; Nilsson, Jonas A

    2016-04-26

    Cell line-derived xenografts (CDXs) are an integral part of drug efficacy testing during development of new pharmaceuticals against cancer but their accuracy in predicting clinical responses in patients have been debated. Patient-derived xenografts (PDXs) are thought to be more useful for predictive biomarker identification for targeted therapies, including in metastatic melanoma, due to their similarities to human disease. Here, tumor biopsies from fifteen patients and ten widely-used melanoma cell lines were transplanted into immunocompromised mice to generate PDXs and CDXs, respectively. Gene expression profiles generated from the tumors of these PDXs and CDXs clustered into distinct groups, despite similar mutational signatures. Hypoxia-induced gene signatures and overexpression of the hypoxia-regulated miRNA hsa-miR-210 characterized CDXs. Inhibition of hsa-miR-210 with decoys had little phenotypic effect in vitro but reduced sensitivity to MEK1/2 inhibition in vivo, suggesting down-regulation of this miRNA could result in development of resistance to MEK inhibitors.

  10. Cytotoxic drug sensitivity of Epstein-Barr virus transformed lymphoblastoid B-cells

    Directory of Open Access Journals (Sweden)

    Olah Eva

    2006-11-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV is the causative agent of immunosuppression associated lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD, AIDS related immunoblastic lymphomas (ARL and immunoblastic lymphomas in X-linked lymphoproliferative syndrome (XLP. The reported overall mortality for PTLD often exceeds 50%. Reducing the immunosuppression in recipients of solid organ transplants (SOT or using highly active antiretroviral therapy in AIDS patients leads to complete remission in 23–50% of the PTLD/ARL cases but will not suffice for recipients of bone marrow grafts. An additional therapeutic alternative is the treatment with anti-CD20 antibodies (Rituximab or EBV-specific cytotoxic T-cells. Chemotherapy is used for the non-responding cases only as the second or third line of treatment. The most frequently used chemotherapy regimens originate from the non-Hodgkin lymphoma protocols and there are no cytotoxic drugs that have been specifically selected against EBV induced lymphoproliferative disorders. Methods As lymphoblastoid cell lines (LCLs are well established in vitro models for PTLD, we have assessed 17 LCLs for cytotoxic drug sensitivity. After three days of incubation, live and dead cells were differentially stained using fluorescent dyes. The precise numbers of live and dead cells were determined using a custom designed automated laser confocal fluorescent microscope. Results Independently of their origin, LCLs showed very similar drug sensitivity patterns against 29 frequently used cytostatic drugs. LCLs were highly sensitive for vincristine, methotrexate, epirubicin and paclitaxel. Conclusion Our data shows that the inclusion of epirubicin and paclitaxel into chemotherapy protocols against PTLD may be justified.

  11. Derivation of the human embryonic stem cell line RCe006-A (RC-2

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe006-A (RC-2 was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12. Microsatellite DNA marker identity and HLA and blood group typing data are available.

  12. Derivation of the human embryonic stem cell line RCe014-A (RC-10

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe014-A (RC-10 was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  13. Derivation of the human embryonic stem cell line RCe012-A (RC-8

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe012-A (RC-8 was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  14. Derivation of the human embryonic stem cell line RCe011-A (RC-7

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe011-A (RC-7 was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  15. Derivation of the human embryonic stem cell line RCe010-A (RC-6

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe010-A (RC-6 was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  16. Effects of chitin and its derivatives on human cancer cells lines.

    Science.gov (United States)

    Bouhenna, M; Salah, R; Bakour, R; Drouiche, N; Abdi, N; Grib, H; Lounici, H; Mameri, N

    2015-10-01

    The present study is focused on the effect of chitin derivatives against human cancer cell lines RD and Hep2. As an outcome from this research, chitin was cytotoxic at IC50 = 400 μg/ml and 200 μg/ml against Hep2 cells and RD cells lines, respectively. Irradiated chitin had an IC50 value of 450 μg/ml for Hep2 and an IC50 of 200 μg/ml for RD. The lowest IC50 is attributed to chitosan, 300 μg/ml in Hep2 and 190 μg/ml in RD.

  17. Established preblastocyst- and blastocyst-derived ES cell lines have highly similar gene expression profiles, despite their differing requirements for derivation culture conditions.

    Science.gov (United States)

    Kim, Chul; Park, Joonghoon; Amano, Tomokazu; Xu, Ren-He; Lin, Ge; Carter, Mark G; Tian, Xiuchun Cindy

    2012-02-01

    The efficiency of embryonic stem (ES) cell derivation relies on an optimized culture medium and techniques for treating preimplantation stage embryos. Recently, ES cell derivation from the preblastocyst developmental stage was reported by removing the zona pellucida from embryos of the most efficient strain for ES cell derivation (129Sv) during early preimplantation. Here, we showed that ES cells can be efficiently derived and maintained in a modified medium (MEMα), from preblastocysts of a low-efficiency mouse strain (a hybrid consisting of 50% B6, 25% CBA, and 25% DBA). Preblastocyst-derived ES cell lines were normal in terms of pluripotency-related protein expression, and chromosome number. Also, preblastocyst-derived ES cell lines from various culture conditions showed pluripotency in vivo through teratoma analysis. Interestingly, ES cell lines produced from preblastocysts and blastocysts, regardless of the derivation culture conditions, are nearly indistinguishable by their global gene expression profiles.

  18. Synthesis of indazole based diarylurea derivatives and their antiproliferative activity against tumor cell lines.

    Science.gov (United States)

    Zhao, Cui-rong; Wang, Rui-qi; Li, Gang; Xue, Xiao-xia; Sun, Chang-jun; Qu, Xian-jun; Li, Wen-bao

    2013-04-01

    New series of indazole based diarylureas were synthesized and their anticancer activity against cancer cells H460, A549, OS-RC-2, HT-29, Lovo, HepG2, Bel-7402, SGC-7901 and MDA-MB-231 were examined. These derivatives of diarylureas, except azaindazole based diarylureas 5f, 5l and 5m, showed superior or similar activity against most of these selected cancer cell lines to the reference compound sorafenib. The effect of substituents on the indazole ring was also investigated. Derivatives with trifluoromenthy or halogen substituent on the indazole ring showed higher activity against the selected cancer cell lines than sorafenib. The acute toxicity assay showed that compounds 5a, 5b and 5i possessed lower toxicity than sorafenib. Compound 5i with 4-(trifluoromenthy)-1H-indazole and 4-(trifluoromenthy) benzene moieties exhibited the most potent anticancer activity.

  19. Comprehensive characterization of genomic instability in pluripotent stem cells and their derived neuroprogenitor cell lines

    Directory of Open Access Journals (Sweden)

    Nestor Luis Lopez Corrales

    2012-12-01

    Full Text Available The genomic integrity of two human pluripotent stem cells and their derived neuroprogenitor cell lines was studied, applying a combination of high-resolution genetic methodologies. The usefulness of combining array-comparative genomic hybridization (aCGH and multiplex fluorescence in situ hybridization (M-FISH techniques should be delineated to exclude/detect a maximum of possible genomic structural aberrations. Interestingly, in parts different genomic imbalances at chromosomal and subchromosomal levels were detected in pluripotent stem cells and their derivatives. Some of the copy number variations were inherited from the original cell line, whereas other modifications were presumably acquired during the differentiation and manipulation procedures. These results underline the necessity to study both pluripotent stem cells and their differentiated progeny by as many approaches as possible in order to assess their genomic stability before using them in clinical therapies.

  20. Stability Test For Sorghum Mutant Lines Derived From Induced Mutations with Gamma-Ray Irradiation

    Directory of Open Access Journals (Sweden)

    S. Human

    2011-12-01

    Full Text Available Sorghum breeding program had been conducted at the Center for the Application of Isotopes and Radiation Technology, BATAN. Plant genetic variability was increased through induced mutations using gamma-ray irradiation. Through selection process in successive generations, some promising mutant lines had been identified to have good agronomic characteristics with high grain yield. These breeding lines were tested in multi location trials and information of the genotypic stability was obtained to meet the requirements for officially varietal release by the Ministry of Agriculture. A total of 11 sorghum lines and varieties consisting of 8 mutant lines derived from induced mutations (B-100, B-95, B-92, B-83, B-76, B-75, B-69 and Zh-30 and 3 control varieties (Durra, UPCA-S1 and Mandau were included in the experiment. All materials were grown in 10 agro-ecologically different locations namely Gunungkidul, Bantul, Citayam, Garut, Lampung, Bogor, Anyer, Karawaci, Cianjur and Subang. In each location, the local adaptability test was conducted by randomized block design with 3 replications. Data of grain yield was used for evaluating genotypic stability using AMMI approach. Results revealed that sorghum mutation breeding had generated 3 mutant lines (B-100, B-76 and Zh-30 exhibiting grain yield significantly higher than the control varieties. These mutant lines were genetically stable in all locations so that they would be recommended for official release as new sorghum varieties to the Ministry of Agriculture

  1. SCREENING FOR CUCUMBER MOSAIC RESISTANT LINES FROM THE OVULE CULTURE DERIVED DOUBLE HAPLOID CUCUMBERS

    Directory of Open Access Journals (Sweden)

    Parichat Plapung

    2014-01-01

    Full Text Available CMV is one of the major destructive viruses worldwide and commercial CMV resistant cucumber is very rare. Therefore we aimed to establish the ovule derived resistant lines used for the breeding program. Haploid plants of sixty-eight cucumber lines were successfully obtained by culturing the un pollinated ovaries harvested one day before an thesis on a modified MS medium supplemented with BAP and IAA at the ratio of 2:1 which was optimal to induce embryogenesis in most of the tested lines. For whole plant regeneration, another modified MS medium was used supplemented with a combination of 6-Benzylaminopurine (BAP and Indole-3-Acetic Acid (IAA (2:1 or BAP and IAA/6-(gamma, gamma-dimethylallylamino purine (2ip and IAA (5:1 and 5 ppm AgNO3. Ploidy levels of the regenerants were determined by cytological analysis. Thirteen out of 42 clones derived from 14 accessions showed a chromosome number of n = 7 and chloroplast number of 6/pair of guard cell, 24 lines were auto-dihaploid with n = 14 and a chloroplast number of 11-12/pair of guard cell. Twenty-eight Double Haploid (DH lines were mechanically inoculated with CMV and the level of resistance was evaluated by using DAS-ELISA. Ten highly Resistant lines (R included 70S2, 91e, 91.1, 93S4-1, 93S42-, 95S1-2, 95S2 DHS1, 117S2-1-3, 136.1 and 194S1 did not show any virus symptom and gave negative ELISA results. Twelve moderately resistant clones were identified including two clones from line 11, three clones from line 93, four clones from line 91 and one clone each from line 117S2 and 123, whereas clone 11.4 was moderately susceptible. Five DH clones; 117S2-1-1, 117S2-2, 117S2-4, 117S2-7 and 117S2-8were classified as highly susceptible.

  2. Derivation of Trisomy 21 affected human embryonic stem cell line Genea021.

    Science.gov (United States)

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Main, Heather; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1-60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. Copyright © 2016 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  3. Derivation of NEM2 affected human embryonic stem cell line Genea080

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea080 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male allele pattern. The hESC line had pluripotent cell morphology, 90% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 99% SSEA4 and gave a PluriTest Pluripotency score of 32.08, Novelty of 1.3. The cell line was negative for Mycoplasma and visible contamination.

  4. Derivation of NEM2 affected human embryonic stem cell line Genea079

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

  5. Derivation of NEM2 affected human embryonic stem cell line Genea078

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea078 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 76% of cells expressed Nanog, 93% Oct4, 67% Tra1–60 and 97% SSEA4 and gave a Pluritest Pluripotency score of 42.18, Novelty of 1.37. The cell line was negative for Mycoplasma and visible contamination.

  6. Establishment and cryopreservation of a giant panda skeletal muscle-derived cell line.

    Science.gov (United States)

    Yu, Fang-Jian; Zeng, Chang-Jun; Zhang, Yan; Wang, Cheng-Dong; Xiong, Tie-Yi; Fang, Sheng-Guo; Zhang, He-Min

    2015-06-01

    The giant panda Ailuropoda melanoleuca is an endangered species and is a symbol for wildlife conservation. Although efforts have been made to protect this rare and endangered species through breeding and conservative biology, the long-term preservation of giant panda genome resources (gametes, tissues, organs, genomic libraries, etc.) is still a practical option. In this study, the giant panda skeletal muscle-derived cell line was successfully established via primary explants culture and cryopreservation techniques. The population doubling time of giant panda skeletal cells was approximately 33.8 h, and this population maintained a high cell viability before and after cryopreservation (95.6% and 90.7%, respectively). The two skeletal muscle-specific genes SMYD1 and MYF6 were expressed and detected by RT-PCR in the giant panda skeletal muscle-derived cell line. Karyotyping analysis revealed that the frequencies of giant panda skeletal muscle cells showing a chromosome number of 2n=42 ranged from 90.6∼94.2%. Thus, the giant panda skeletal muscle-derived cell line provides a vital resource and material platform for further studies and is likely to be useful for the protection of this rare and endangered species.

  7. Derivation and characterization of human embryonic stem cell lines from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhao Wu; Huimin Dai; Lei Qian; Qing Tian; Lei Xiao; Xiaojun Tan; Hui Li; Lingjun Rao; Lixiazi He; Lei Bao; Jing Liao; Chun Cui; Zhenyu Zuo; Qiao Li

    2011-01-01

    Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.

  8. Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

    Directory of Open Access Journals (Sweden)

    Svetlana Gavrilov

    2011-01-01

    Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

  9. Arsenite-induced autophagy is associated with proteotoxicity in human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Bolt, Alicia M.; Zhao, Fei; Pacheco, Samantha; Klimecki, Walter T., E-mail: klimecki@pharmacy.arizona.edu

    2012-10-15

    Epidemiological studies of arsenic-exposed populations have provided evidence that arsenic exposure in humans is associated with immunosuppression. Previously, we have reported that arsenite-induced toxicity is associated with the induction of autophagy in human lymphoblastoid cell lines (LCL). Autophagy is a cellular process that functions in the degradation of damaged cellular components, including protein aggregates formed by misfolded or damaged proteins. Accumulation of misfolded or damaged proteins in the endoplasmic reticulum (ER) lumen causes ER stress and activates the unfolded protein response (UPR). In an effort to investigate the mechanism of autophagy induction by arsenite in the LCL model, we examined the potential contribution of ER stress and activation of the UPR. LCL exposed to sodium arsenite for 8-days induced expression of UPR-activated genes, including CHOP and GRP78, at the RNA and the protein level. Evidence for activation of the three arms of the UPR was observed. The arsenite-induced activation of the UPR was associated with an accumulation of protein aggregates containing p62 and LC3, proteins with established roles in the sequestration and autophagic clearance of protein aggregates. Taken together, these data provide evidence that arsenite-induced autophagy is associated with the generation of ER stress, activation of the UPR, and formation of protein aggregates that may be targeted to the lysosome for degradation. -- Highlights: ► Arsenite induces endoplasmic reticulum stress and the unfolded protein response. ► Arsenite induces the formation of protein aggregates that contain p62 and LC3-II. ► Time-course data suggests that arsenite-induced autophagy precedes ER stress.

  10. Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines

    Directory of Open Access Journals (Sweden)

    Vázquez Guelaguetza

    2003-03-01

    Full Text Available Abstract Background Uterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH experiments have detected a specific pattern of chromosomal imbalances during cervical cancer progression, indicating chromosomal regions that might contain genes that are important for cervical transformation. Methods We performed HPV detection and CGH analysis in order to initiate the genomic characterization of four recently established cervical carcinoma derived cell lines from Mexican patients. Results All the cell lines were HPV18 positive. The most prevalent imbalances in the cell lines were gains in chromosomes 1q23-q32, 3q11.2-q13.1, 3q22-q26.1, 5p15.1-p11.2, this alteration present as a high copy number amplification in three of the cell lines, 7p15-p13, 7q21, 7q31, 11q21, and 12q12, and losses in 2q35-qter, 4p16, 6q26-qter, 9q34 and 19q13.2-qter. Conclusions Analysis of our present findings and previously reported data suggest that gains at 1q31-q32 and 7p13-p14, as well as losses at 6q26-q27 are alterations that might be unique for HPV18 positive cases. These chromosomal regions, as well as regions with high copy number amplifications, coincide with known fragile sites and known HPV integration sites. The general pattern of chromosomal imbalances detected in the cells resembled that found in invasive cervical tumors, suggesting that the cells represent good models for the study of cervical carcinoma.

  11. Deriving precise parameters for cool solar-type stars. Optimizing the iron line list

    Science.gov (United States)

    Tsantaki, M.; Sousa, S. G.; Adibekyan, V. Zh.; Santos, N. C.; Mortier, A.; Israelian, G.

    2013-07-01

    Context. Temperature, surface gravity, and metallicitity are basic stellar atmospheric parameters necessary to characterize a star. There are several methods to derive these parameters and a comparison of their results often shows considerable discrepancies, even in the restricted group of solar-type FGK dwarfs. Aims: We want to check the differences in temperature between the standard spectroscopic technique based on iron lines and the infrared flux method (IRFM). We aim to improve the description of the spectroscopic temperatures especially for the cooler stars where the differences between the two methods are higher, as presented in a previous work. Methods: Our spectroscopic analysis was based on the iron excitation and ionization balance, assuming Kurucz model atmospheres in LTE. The abundance analysis was determined using the code MOOG. We optimized the line list using a cool star (HD 21749) with high resolution and high signal-to-noise spectrum, as a reference in order to check for weak, isolated lines. Results: We test the quality of the new line list by re-deriving stellar parameters for 451 stars with high resolution and signal-to-noise HARPS spectra, that were analyzed in a previous work with a larger line list. The comparison in temperatures between this work and the latest IRFM for the stars in common shows that the differences for the cooler stars are significantly smaller and more homogeneously distributed than in previous studies for stars with temperatures below 5000 K. Moreover, a comparison is presented between interferometric temperatures with our results that shows good agreement, even though the sample is small and the errors of the mean differences are large. We use the new line list to re-derive parameters for some of the cooler stars that host planets. Finally, we present the impact of the new temperatures on the [Cr i/Cr ii] and [Ti i/Ti ii] abundance ratios that previously showed systematic trends with temperature. We show that the slopes

  12. Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

    Science.gov (United States)

    Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

    2010-04-01

    The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

  13. Design, synthesis and cytotoxicity studies of dithiocarbamate ester derivatives of emetine in prostate cancer cell lines.

    Science.gov (United States)

    Akinboye, Emmanuel S; Bamji, Zebalda D; Kwabi-Addo, Bernard; Ejeh, David; Copeland, Robert L; Denmeade, Samuel R; Bakare, Oladapo

    2015-09-01

    A small library of emetine dithiocarbamate ester derivatives were synthesized in 25-86% yield via derivatization of the N2'- position of emetine. Anticancer evaluation of these compounds in androgen receptor positive LNCaP and androgen receptor negative PC3 and DU145 prostate cancer cell lines revealed time dependent and dose-dependent cytotoxicity. With the exception of compound 4c, all the dithiocarbamate ester analogs in this study showed appreciable potency in all the prostate cancer cell lines (regardless of whether it is androgen receptor positive or negative) with a cytotoxicity IC50 value ranging from 1.312 ± 0.032 μM to 5.201 ± 0.125 μM by day 7 of treatment. Compared to the sodium dithiocarbamate salt 1, all the dithiocarbamate ester analogs (2 and 4a-4 g) displayed lower cytotoxicity than compound 1 (PC3, IC50 = 0.087 ± 0.005 μM; DU145, IC50 = 0.079 ± 0.003 μM and LNCaP, IC50 = 0.079 ± 0.003 μM) on day 7 of treatment. Consequently, it appears that S-alkylation of compound 1 leads to a more stable dithiocarbamate ester derivative that resulted in lower anticancer activity in the prostate cancer cell lines.

  14. Effect of proteasome inhibitors on proliferation and apoptosis of human cutaneous melanoma-derived cell lines.

    Science.gov (United States)

    Sorolla, A; Yeramian, A; Dolcet, X; Pérez de Santos, A M; Llobet, D; Schoenenberger, J A; Casanova, J M; Soria, X; Egido, R; Llombart, A; Vilella, R; Matias-Guiu, X; Marti, R M

    2008-03-01

    Cutaneous malignant melanoma is an aggressive type of skin cancer which causes disproportionate mortality in young and middle-aged adults. Once disseminated, melanoma can be considered an incurable disease, highly resistant to standard antineoplastic treatment, such as chemotherapy or radiation therapy. The proteasome represents a novel target for cancer therapy that can potentially be used in melanoma. To assess the effect of four structurally different proteasome inhibitors on human cutaneous melanoma-derived cell lines. Sixteen human cutaneous melanoma-derived cell lines which are original were obtained from patients who were treated by two of the authors. Cells were cultured, exposed to proteasome inhibitors (bortezomib, ALLN, MG-132 and epoxomicin) and then assayed for cell cycle and cell death analyses. Proteasome inhibitors inhibited the in vitro growth of melanoma cells, and this effect was due to a reduction in cell proliferation rate and an induction of both caspase-dependent and caspase-independent cell death. Moreover, release of apoptosis-inducing factor was observed in the presence of the broad-specificity caspase inhibitor BAF (Boc-D-fmk). In addition, the four different proteasome inhibitors induced caspase 2 processing. This study provides information regarding the in vitro effects of proteasome inhibitors on melanoma cell lines, and the molecular mechanisms involved. It also gives support to the future use of such inhibitors in the treatment of patients with melanoma, either administered alone or in combination with other drugs.

  15. A General Equation Derived by Field Theory for the Use of Uniform Transmission Line with Mutual Inductance

    Institute of Scientific and Technical Information of China (English)

    HONGQingquan; WANGJiancheng; CHENShennian

    2004-01-01

    Beginning with 2 independent equations in integral form derived from Maxwell equation, a most general equation is inferred for the use of uniform transmission line where mutual inductance exists between two conductors in all different line segments of unit lengths;and moreover, the formulas of the magnitude of parameters are given for the use of line segments of unit length on uniform transmission line. A method of field theory is thus offered for analyzing distributed parameter circuit.

  16. Deriving precise parameters for cool solar-type stars. Optimizing the iron line list

    CERN Document Server

    Tsantaki, M; Adibekyan, V Zh; Santos, N C; Mortier, A; Israelian, G

    2013-01-01

    Temperature, surface gravity, and metallicitity are basic stellar atmospheric parameters necessary to characterize a star. We aim to improve the description of the spectroscopic temperatures especially for the cooler stars where the differences with the Infrared Flux Method are higher, as presented in previous work. Our spectroscopic analysis is based on the iron excitation and ionization balance, assuming Kurucz model atmospheres in LTE. The abundance analysis is determined using the code MOOG. We optimize the line list using a cool star with high resolution and high signal-to-noise spectrum, as a reference in order to check for weak, isolated lines. We test the quality of the new line list by re-deriving stellar parameters for 451 stars with high resolution and signal-to-noise HARPS spectra, that were analyzed in a previous work with a larger line list. The comparison in temperatures between this work and the latest IRFM shows that the differences for the cooler stars are significantly smaller and more homo...

  17. Pharmacokinetics of intravitreal glial cell line-derived neurotrophic factor: experimental studies in pigs

    DEFF Research Database (Denmark)

    Ejstrup, Rasmus; Kiilgaard, J F; Tucker, B A;

    2010-01-01

    The purpose of this study was to establish the intravitreal (ITV) pharmacokinetics of glial cell line-derived neurotrophic factor (GDNF) and observe possible complications after ITV injection. Twenty Danish landrace pigs and 34 eyes were included in the study; 30 were injected with 100 ng of GDNF......, two controls were injected without GDNF, and two received no injection. At post-injection time points of 1, 2, 3, 6 hours (h), 1, 2, 4 or 7 days (d) eyes were enucleated and the ITV concentration of GDNF (cGDNF) was determined by enzyme-linked immunosorbent assay, and activity was tested using...... a retinal ganglion cell line (RGC5) bioassay. Indirect ophthalmoscopy, intraocular pressure assessment, and fundus photography were performed before enucleation. There was initial variability in the cGDNF, but after 24h GDNF was cleared in a monoexponential fashion with a half-life of 37 h (CL 33-43 h...

  18. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

    Science.gov (United States)

    Stanga, Serena; Zanou, Nadège; Audouard, Emilie; Tasiaux, Bernadette; Contino, Sabrina; Vandermeulen, Gaëlle; René, Frédérique; Loeffler, Jean-Philippe; Clotman, Frédéric; Gailly, Philippe; Dewachter, Ilse; Octave, Jean-Noël; Kienlen-Campard, Pascal

    2016-05-01

    Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.

  19. Synthesis and Biological Evaluation of Lipophilic 1,4-Naphthoquinone Derivatives against Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Shao-Hung Wang

    2015-06-01

    Full Text Available To examine the effect of hydrophobicity on the anticancer activity of 1,4-naphthoquinone derivatives, a series of compounds bearing a 2-O-alkyl-, 3-C-alkyl- or 2/3-N-morpholinoalkyl group were synthesized and evaluated for their anticancer activity against five human cancer cell lines in vitro. The cytotoxicity of these derivatives was assayed against HT-29, SW480, HepG2, MCF-7 and HL-60 cells by the MTT assay. Among them, 2-hydroxy-3-farnesyl-1,4-naphthoquinone (11a was found to be the most cytotoxic against these cell lines. Our results showed that the effectiveness of compound 11a may be attributed to its suppression of the survival of HT-29. Secondly, in the Hoechst 33258 staining test, compound 11a-treated cells exhibited nuclear condensation typical of apoptosis. Additionally, cell cycle analysis by flow cytometry indicated that compound 11a arrested HT-29 cells in the S phase. Furthermore, cell death detected by Annexin V-FITC/propidium iodide staining showed that compound 11a efficiently induced apoptosis of HT-29 in a concentration-dependent manner. Taken together, compound 11a effectively inhibits colon cancer cell proliferation and may be a potent anticancer agent.

  20. Data-Derived Estimation of Source Depth Using Vertical Line Array Data in Shallow Water

    Institute of Scientific and Technical Information of China (English)

    WANG Hao-Zhong; WANG Ning; GAO Da-Zhi

    2011-01-01

    Following the closure relation of normal mode theory, the source depth can be estimated approximately provided that the eigenfunction and the excited amplitude with correct polarity of each effective mode are given. Both the eigenfunction and the excited amplitude of each effective mode can be extracted using the data-derived method, which does not require a priori knowledge about ocean environment and source range but the sound pressure data observed by vertical line array. The polarities, undetermined by data-derived method, can be estimated using a cost function. Then, the source depth is determined by locating the peak of the object function defined by the closure relation with the correct polarities. Numerical simulation and experiment are carried out to illustrate the performance of this method.%Following the closure relation of normal mode theory,the source depth can be estimated approximately provided that the eigenfunction and the excited amplitude with correct polarity of each effective mode are given.Both the eigenfunction and the excited amplitude of each effective mode can be extracted using the data-derived method,which does not require a priori knowledge about ocean environment and source range but the sound pressure data observed by vertical line array.The polarities,undetermined by data-derived method,can be estimated using a cost function.Then,the source depth is determined by locating the peak of the object function defined by the closure relation with the correct polarities.Numerical simulation and experiment are carried out to illustrate the performance of this method.Long-range source depth estimation using a vertical line array (VLA) is desirable in practice.By finding the peak in the correlation function between the modeled acoustic field (for an assumed source depth) and the received pressure field at a VLA,the matched field processing technique (MFP) can be used to estimate source depth.[1-3] Assuming that the pressure field can be

  1. Cryptic introgression of Dasypyrum villosum parental DNA in wheat lines derived from intergeneric hybridization.

    Science.gov (United States)

    Caceres, M E; Pupilli, F; Ceccarelli, M; Vaccino, P; Sarri, V; De Pace, C; Cionini, P G

    2012-01-01

    Cytogenetic and DNA molecular analyses have been carried out in 3 wheat introgression lines (ILs; CS×V58, CS×V59, and CS×V60) derived from Triticum aestivum cv. 'Chinese Spring' (CS) × Dasypyrum villosum(Dv) intergeneric hybridization. All lines, which showed several phenotypic differences compared to CS, had the same chromosome number (2n = 42) and structure as CS, and neither chromosomes nor chromatin from Dv were apparently added to their complement. However, Feulgen/DNA cytophotometry showed that there was more nuclear DNA in the lines than in the parental wheat (by 1.85%, 2.76%, and 1.26% in CS×V58, CS×V59, and CS×V60, respectively). Molecular investigation indicated the presence of Dv DNA in the ILs. AFLP analysis of genomic DNA from the ILs, CS, and Dv detected a total of 120 polymorphic bands, of which 7 (5.8%) were present in some or all the ILs and Dv but were absent in CS. PCR amplification, sequence analysis of amplicons, and Southern blot hybridization confirmed the presence of Dv-specific sequences in each of the ILs. These results indicate cryptic introgression of Dv DNA sequences into the genome of the ILs. Some implications of this finding are discussed. Copyright © 2011 S. Karger AG, Basel.

  2. Effect of glial cell line-derived neurotrophic factor on retinal function after experimental branch retinal vein occlusion

    DEFF Research Database (Denmark)

    Ejstrup, Rasmus; Dornonville de la Cour, Morten; Kyhn, Maria Voss;

    2012-01-01

    The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs.......The objective of the study was to investigate the effect of glial cell line-derived neurotrophic factor (GDNF) on the multifocal electroretinogram (mfERG) following an induced branch retinal vein occlusion (BRVO) in pigs....

  3. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.

    Science.gov (United States)

    Zou, Chunlin; Chou, Bin-Kuan; Dowey, Sarah N; Tsang, Kitman; Huang, Xiaosong; Liu, Cyndi F; Smith, Cory; Yen, Jonathan; Mali, Prashant; Zhang, Yu Alex; Cheng, Linzhao; Ye, Zhaohui

    2012-08-10

    Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

  4. Synthesis and cytotoxic activity of certain benzothiazole derivatives against human MCF-7 cancer cell line.

    Science.gov (United States)

    Mohamed, Lamia W; Taher, Azza T; Rady, Ghada S; Ali, Mamdouh M; Mahmoud, Abeer E

    2016-10-04

    A new series of benzothiazole has been synthesized as cytotoxic agents. The new derivatives were tested for their cytotoxic activity toward the human breast cancer MCF-7 cell line against cisplatin as the reference drug. Many derivatives revealed good cytotoxic effect, whereas four of them, 4, 5c, 5d, and 6b, were more potent than cisplatin, with IC50 values being 8.64, 7.39, 7.56, and 5.15 μm compared to 13.33 μm of cisplatin. The four derivatives' cytotoxic activity was accompanied by regulating free radicals production, by increasing the activity of superoxide dismutase and depletion of intracellular reduced glutathione, catalase, and glutathione peroxidase activities, accordingly, the high production of hydrogen peroxide, nitric oxide, and other free radicals causing tumor cell death as monitored by reduction in the synthesis of protein and nucleic acids. Most of the tested compounds showed potent to moderate growth inhibitory activity; in particular, compound 6b exhibited the highest activity suggesting it is a lead compound in cytotoxic activity.

  5. Exposure to cobalt causes transcriptomic and proteomic changes in two rat liver derived cell lines.

    Science.gov (United States)

    Permenter, Matthew G; Dennis, William E; Sutto, Thomas E; Jackson, David A; Lewis, John A; Stallings, Jonathan D

    2013-01-01

    Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.

  6. Exposure to cobalt causes transcriptomic and proteomic changes in two rat liver derived cell lines.

    Directory of Open Access Journals (Sweden)

    Matthew G Permenter

    Full Text Available Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.

  7. Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

    Science.gov (United States)

    Permenter, Matthew G.; Dennis, William E.; Sutto, Thomas E.; Jackson, David A.; Lewis, John A.; Stallings, Jonathan D.

    2013-01-01

    Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies. PMID:24386269

  8. S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk.

    Directory of Open Access Journals (Sweden)

    Ruisong Li

    Full Text Available BACKGROUND: Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF, and glial cell line-derived neurotrophic factor (GDNF in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05. In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05. Delivery modes were negatively associated with the concentration of GDNF in human milk. CONCLUSIONS: S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.

  9. Molecular Characterization of Atlas 66-Derived Wheat Near-Isogenic Lines Contrasting in Aluminum (Al) Tolerance

    Institute of Scientific and Technical Information of China (English)

    GUO Pei-guo; BAI Gui-hua; LI Rong-hua; Brett Carver; Michael Baum

    2007-01-01

    Aluminum (Al) toxicity is the major limiting factor for wheat growth in acidic soils. Genetic improvement of Al tolerance is one of the most cost-effective solutions to improve wheat productivity. The objective of this study was to characterize near isogenic lines (NILs) contrasting in Al tolerance derived from Atlas 66 in the backgrounds of Al-sensitive cultivars Chisholm and Century using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR). A total of 200 AFLP and 88 SSR primer pairs were screened and 12 markers (11 AFLPs and one SSR) were associated with Altolerance in NILs of at least one recurrent parental background. Among them, nine were linked to Al tolerance in the Chisholm-derived NILs, seven were associated with Al-tolerance in the Century-derived NILs, and three AFLPs derived from the primer combinations of pAG/mGCAG, pCAG/mAGC and pGTG/mGCG, and one SSR, Xwmc331 on chromosome4D, associated with Al tolerance in NILs of both recurrent parental backgrounds. Those common markers across two backgrounds may be the major marker loci associated with Al-tolerance in Atlas 66 and could be useful for marker-assisted breeding to improve Al tolerance in wheat. In addition, evaluation of Al tolerance among different genotypes using hematoxylin stain and relative root growth revealed that Atlas 66 was more tolerant to Al stress than the NILs, therefore suggested that the Al-tolerant NILs might not carry all Al-tolerance loci from Atlas 66 and inheritance of Al tolerance in Atlas 66 is more likely multigenic.

  10. Postnatal roles of glial cell line-derived neurotrophic factor family members in nociceptors plasticity

    Institute of Scientific and Technical Information of China (English)

    Sacha A. Malin; Brian M. Davis

    2008-01-01

    The neurotrophin and glial cell line-derived neurotrophic factor (GDNF) family of growth factors have been extensively studied because of their proven ability to regulate development of the peripheral nervous system. The neurotrophin family,which includes nerve growth factor (NGF), NT-3, NT4/5 and BDNF, is also known for its ability to regulate the function of adult sensory neurons. Until recently, little was known concerning the role of the GNDF-family (that includes GDNF, artemin, neurturin and persephin) in adult sensory neuron function. Here we describe recent data that indicates that the GDNF family can regulate sensory neuron function, that some of its members are elevated in inflammatory pain models and that application of these growth factors produces pain in vivo. Finally we discuss how these two families of growth factors may converge on a single membrane receptor, TRPV 1, to produce long-lasting hyperalgesia.

  11. Effect of glial cell line-derived neurotrophic factor on peripheral nerve regeneration in adult rat

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhe-yu; LI Jian-hong; ZHENG Xing-dong; LU Chang-lin; HE Cheng

    2001-01-01

    Objective: To study the effect of glial cell line-derived neurotrophic (GDNF) on adult peripheral nerve regeneration. Methods: Transectioned sciatic nerve in adult rats was sutured into silicone channel. GDNF or SAL solution was injected into the silicone channels during operation. Four weeks later, the effect of GDNF on axonal regeneration was evaluated by degenerative neurofiber staining and HRP retrograde tracing. Results: Compared with SAL group, the percentage of degenerative neurofiber areas decreased from 17.3% to 1.9% ( P<0.01 ) and the ratio of labeled spinal somas number was significantly increased from 43.5% to 68.3% ( P<0.01 ) in GDNF group. Conclusion: The results suggest that exogenous GDNF can obviously enhance adult peripheral nerve regeneration.

  12. Glial cell line-derived neurotrophic factor (GDNF therapy for Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Shingo,Tetsuro

    2007-04-01

    Full Text Available Many studies using animals clarify that glial cell line-derived neurotrophic factor (GDNF has strong neuroprotective and neurorestorative effects on dopaminergic neurons. Several pilot studies clarified the validity of continuous intraputaminal GDNF infusion to patients with Parkinson's disease (PD, although a randomized controlled trial of GDNF therapy published in 2006 resulted in negative outcomes, and controversy remains about the efficacy and safety of the treatment. For a decade, our laboratory has investigated the efficacy and the most appropriate method of GDNF administration using animals, and consequently we have obtained some solid data that correspond to the results of clinical trials. In this review, we present an outline of our studies and other key studies related to GDNF, the current state of the research, problems to be overcome, and predictions regarding the use of GDNF therapy for PD in the future.

  13. Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures

    DEFF Research Database (Denmark)

    Aaberg-Jessen, Charlotte; Nørregaard, Annette; Christensen, Karina

    2013-01-01

    preserving the invasive features and stem cell features of glioma cells. Fluorescently labelled primary glioma spheroids and U87MG cell line-derived spheroids were implanted into organotypic rat corticostriatal slice cultures and the invasion was followed over time by confocal microscopy. The invasion......Gliomas are highly invasive tumors and the pronounced invasive features of gliomas prevent radical surgical resection. In the search for new therapeutics targeting invasive glioma cells, in vivo-like in vitro models are of great interest. We developed and evaluated an in vivo-like in vitro model...... was validated immunohistochemically with paraffin sections using a human-specific vimentin antibody. Moreover, the preservation of immature stem cell features was evaluated immunohistochemically using the stem cell markers CD133, Sox2, Bmi-1 and nestin. The confocal and immunohistochemical results showed...

  14. Human Umbilical Cord Blood-Derived Neural Stem Cell Line as a Screening Model for Toxicity.

    Science.gov (United States)

    Patnaik, Rajashree; Padhy, Rabindra Nath

    2017-04-01

    The aim was to investigate whether a human neural stem cell (NSC) line derived from human umbilical cord blood (hUCB) can be used for toxicity study. Toxicity of both neurotoxic environmental xenobiotics, methyl mercury chloride (CH3HgCl), lead acetate (CH3COOPb), and chlorpyrifos (CP), and non-neurotoxic insecticide, dichlorvos, as well as non-neurotoxic drugs, theophylline and acetaminophen were assessed. Additionally, differentiation of neuronal and glial cell lines derived from hUCB was elucidated. It was observed that CH3HgCl was more toxic to human NSCs in comparison to CH3COOPb and CP. The minimum inhibitory concentration (MIC) value against NSCs was 3, 10, and 300 mg/L, in each staining process, acridine orange/ethidium bromide (AO/EB) staining, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay, and Hoechst staining, for CH3HgCl, CP, and CH3COOPb, respectively. CH3HgCl had the LC25 value as 10.0, 14.4, and 12.7 mg/L, by staining method mentioned in succession. CP had the LC25 value as 21.9, 23.7, and 18.4 mg/L; similarly, CH3COOPb had LC25 values, successively as 616.9, 719.2, and 890.3 mg/L. LC50 values ranged from 18.2 to 21.7 mg/L for CH3HgCl, 56.4 to 60.2 mg/L for CP, and 1000 to 1460.1 for CH3COOPb. Theophylline, acetaminophen, and dichlorvos had no impact on the viability of NSCs. This work justified that hUCB-NSC model can be used for toxicity study.

  15. QTL mapping under truncation selection in homozygous lines derived from biparental crosses.

    Science.gov (United States)

    Melchinger, Albrecht E; Orsini, Elena; Schön, Chris C

    2012-02-01

    In plant breeding, a large number of progenies that will be discarded later in the breeding process must be phenotyped and marker genotyped for conducting QTL analysis. In many cases, phenotypic preselection of lines could be useful. However, in QTL analyses even moderate preselection can have a significant effect on the power of QTL detection and estimation of effects of the target traits. In this study, we provide exact formulas for quantifying the change of allele frequencies within marker classes, expectations of marker contrasts and the variance of the marker contrasts under truncation selection, for the general case of two QTL affecting the target trait and a correlated trait. We focused on homozygous lines derived at random from biparental crosses. The effects of linkage between the marker and the QTL under selection as well as the effect of selection on a correlated trait can be quantified with the given formulas. Theoretical results clearly show that depending on the magnitude of QTL effects, high selection intensities can lead to a dramatic reduction in power of QTL detection and that approximations based on the infinitesimal model deviate substantially from exact solutions. The presented formulas are valuable for choosing appropriate selection intensity when performing QTL mapping experiments on the data on phenotypically preselected traits and enable the calculation and bias correction of the effects of QTL under selection. Application of our theory to experimental data revealed that selection-induced bias of QTL effects can be successfully corrected.

  16. Synthesis of kaempferide Mannich base derivatives and their antiproliferative activity on three human cancer cell lines.

    Science.gov (United States)

    Nguyen, Van-Son; Shi, Ling; Luan, Fang-Qian; Wang, Qiu-An

    2015-01-01

    Kaempferide (3,5,7-trihydroxy-4'-methoxyflavone, 1), a naturally occurring flavonoid with potent anticancer activity in a number of human tumour cell lines, was first semisynthesized from naringin. Based on Mannich reaction of kaempferide with various secondary amines and formaldehyde, nine novel kaempferide Mannich base derivatives 2-10 were synthesized. The aminomethylation occurred preferentially in the position at C-6 and C-8 of the A-ring of kaempferide. All the synthetic compounds were tested for antiproliferative activity against three human cancer cell lines (Hela, HCC1954, SK-OV-3) by the standard MTT method. The results showed that compounds 1, 2 and 5-10 were more potent against Hela cells with IC50 values of 12.47-28.24 μM than the positive control cis-platin (IC50 41.25 μM), compounds 5, 6, 8 and 10 were more potent against HCC1954 cells with IC50 values of 8.82-14.97 μM than the positive control cis-platin (IC50 29.68 μM), and compounds 2, 3, 5, 6 and 10 were more potent against SK-OV-3 cells with IC50 values of 7.67-18.50 μM than the positive control cis-platin (IC50 21.27 μM).

  17. Characterization of HGF/Met Signaling in Cell Lines Derived From Urothelial Carcinoma of the Bladder

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young H. [Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Apolo, Andrea B. [Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Agarwal, Piyush K.; Bottaro, Donald P., E-mail: dbottaro@helix.nih.gov [Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2014-11-25

    There is mounting evidence of oncogenic hepatocyte growth factor (HGF)/Met signaling in urothelial carcinoma (UC) of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID) mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice vs. SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.

  18. Characterization of HGF/Met Signaling in Cell Lines Derived From Urothelial Carcinoma of the Bladder

    Directory of Open Access Journals (Sweden)

    Young H. Lee

    2014-11-01

    Full Text Available There is mounting evidence of oncogenic hepatocyte growth factor (HGF/Met signaling in urothelial carcinoma (UC of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice vs. SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.

  19. Characterization of HGF/Met Signaling in Cell Lines Derived From Urothelial Carcinoma of the Bladder.

    Science.gov (United States)

    Lee, Young H; Apolo, Andrea B; Agarwal, Piyush K; Bottaro, Donald P

    2014-11-25

    There is mounting evidence of oncogenic hepatocyte growth factor (HGF)/Met signaling in urothelial carcinoma (UC) of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID) mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice vs. SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.

  20. Novel pancreatic cancer cell lines derived from genetically engineered mouse models of spontaneous pancreatic adenocarcinoma: applications in diagnosis and therapy.

    Directory of Open Access Journals (Sweden)

    María P Torres

    Full Text Available Pancreatic cancer (PC remains one of the most lethal human malignancies with poor prognosis. Despite all advances in preclinical research, there have not been significant translation of novel therapies into the clinics. The development of genetically engineered mouse (GEM models that produce spontaneous pancreatic adenocarcinoma (PDAC have increased our understanding of the pathogenesis of the disease. Although these PDAC mouse models are ideal for studying potential therapies and specific genetic mutations, there is a need for developing syngeneic cell lines from these models. In this study, we describe the successful establishment and characterization of three cell lines derived from two (PDAC mouse models. The cell line UN-KC-6141 was derived from a pancreatic tumor of a Kras(G12D;Pdx1-Cre (KC mouse at 50 weeks of age, whereas UN-KPC-960 and UN-KPC-961 cell lines were derived from pancreatic tumors of Kras(G12D;Trp53(R172H;Pdx1-Cre (KPC mice at 17 weeks of age. The cancer mutations of these parent mice carried over to the daughter cell lines (i.e. Kras(G12D mutation was observed in all three cell lines while Trp53 mutation was observed only in KPC cell lines. The cell lines showed typical cobblestone epithelial morphology in culture, and unlike the previously established mouse PDAC cell line Panc02, expressed the ductal marker CK19. Furthermore, these cell lines expressed the epithelial-mesenchymal markers E-cadherin and N-cadherin, and also, Muc1 and Muc4 mucins. In addition, these cell lines were resistant to the chemotherapeutic drug Gemcitabine. Their implantation in vivo produced subcutaneous as well as tumors in the pancreas (orthotopic. The genetic mutations in these cell lines mimic the genetic compendium of human PDAC, which make them valuable models with a high potential of translational relevance for examining diagnostic markers and therapeutic drugs.

  1. Investigation of the apoptotic way induced by digallic acid in human lymphoblastoid TK6 cells

    Directory of Open Access Journals (Sweden)

    Bhouri Wissem

    2012-06-01

    Full Text Available Abstract Background The digallic acid (DGA purified from Pistacia lentiscus. L fruits was investigated for its antiproliferative and apoptotic activities on human lymphoblastoid TK6 cells. Methods We attempt to characterize the apoptotic pathway activated by DGA. Apoptosis was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activities. Results The inhibition of lymphoblastoid cell proliferation was noted from 8.5 μg/ml of DGA. The induction of apoptosis was confirmed by DNA fragmentation and PARP cleavage. We have demonstrated that DGA induces apoptosis by activating the caspase-8 extrinsic pathway. Caspase-3 was also activated in a dose dependent manner. Conclusion In summary, DGA exhibited an apoptosis inductor effect in TK6 cells revealing thus its potential as a cancer-preventive agent.

  2. Properties of interstellar filaments derived from Herschel, Planck, and molecular line observations

    Science.gov (United States)

    Arzoumanian, Doris

    2015-08-01

    The highly filamentary structure of the interstellar medium (ISM) is now impressively revealed by Herschel and Planck images. Previous observations have shown that clouds are filamentary, however, only recently the mapping capabilities of Herscheland Planck have discovered their ubiquity in the ISM. In particular, Herschel images, with their high spatial and intensity dynamic ranges, show that dense filaments are associated with the main sites of star formation, demonstrating their key role in the star formation process.The analysis of the column density profiles of filaments indicates that they all share a common central width of 0.1pc, while they span a wide range in length, column density, mass per unit length. The results derived from observations tracing cold dust and gas emission, in total and polarised intensity, suggest that filaments can be divided into two families: On the one hand, low column density, unbound, and quiescent filaments mostly aligned with the magnetic field orientation, and on the other hand, dense, self-gravitating filaments, which fragment into star forming cores.I will present the properties of the filamentary structures derived from Herschel, Planck, and molecular line observations, and I will discuss the observational constraints on the formation and evolution of interstellar filaments.

  3. Regional estimates of glacier mass change from MODIS-derived equilibrium line altitudes

    Directory of Open Access Journals (Sweden)

    J. M. Shea

    2012-09-01

    Full Text Available We describe an automated method to extract regional snowline elevations and annual equilibrium line altitudes (ELAs from daily MODIS imagery (MOD02QKM on large glaciers and icefields in western North America. Regional MODIS-derived ELAs correlate significantly with observed net mass balance at six index glacier mass balance sites. Historical mass balance gradients were combined with MODIS-derived ELAs to estimate annual mass change at the Columbia, Lillooet, and Sittakanay icefields in British Columbia, Canada. Our approach yields estimates of mass change that are within 30% of traditional geodetic approaches over decadal time-scales, and reveals continued mass loss of glaciers in western North America. Between 2000 and 2009, mean annual rates of surface elevation change for the Columbia, Lillooet, and Sittakanay icefields are estimated to be −0.29 ± 0.15 m a−1, −0.57 ± 0.10 m a−1, and −0.90 ± 0.09 m a−1, respectively. This study provides a complementary approach to the development of regional estimates of glacier mass change, which are critical for studies of glacier contributions to both streamflow and global sea-level rise.

  4. Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells.

    Science.gov (United States)

    Gale, Zoe; Cooper, Paul R; Scheven, Ben A

    2012-02-01

    Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.

  5. New Gravity-Derived Grounding Line Depths Highlight Role Bathymetry Plays in Ongoing Greenland Ice Sheet Change

    Science.gov (United States)

    Boghosian, A.; Porter, D. F.; Tinto, K. J.; Bell, R. E.; Cochran, J. R.; Csatho, B. M.

    2014-12-01

    Bathymetry has been a missing piece in understanding ice-ocean interactions of marine-terminating glaciers in Greenland. As bathymetry in fjords often controls the flow of warm water to the terminus of the glacier, and the grounding line depth of the glacier can modulate the effect of this warm water on the glacier. Study of Tracy and Heilprin Glaciers, a pair of neighboring glaciers in northwest Greenland, has indicated that when exposed to similar external forcings, the glacier with the deeper grounding line will retreat more rapidly. The new comprehensive mapping of grounding line depths with Operation IceBridge gravity inversions provides the basis for examining this question for all of Greenland's glaciers. We consider 54 distinct glaciers in our analysis. Grounding line depths for these 54 come from data collected by Operation IceBridge from 2010-2012. New grounding line depths for 36 glaciers are derived from gravity measurements in locations where radar is unable to retrieve grounding line depths. 18 grounding line depths come from CReSIS Multichannel Coherent Depth Sounder radar data. Offshore bathymetry in all 54 fjords is gravity-derived. The gravity-derived bathymetry is always constrained onshore by radar, and when possible pinned offshore to acoustic bathymetric measurements.Here we present the gravity-derived grounding line depths along with recent glacier behavior, including surface lowering and terminus retreat, of these 54 glaciers. In general, the glaciers with deeper grounding lines are experiencing greater mass loss. This relationship between grounding line depth and mass balance extends to much of Greenland. We systematically discuss this relationship in the different major regions of Greenland, and note that the relationship is strongest in the southeast, and weakest in the north.

  6. Clonal preservation of human pancreatic cell line derived from primary pancreatic adenocarcinoma.

    Science.gov (United States)

    Mohammad, R M; Li, Y; Mohamed, A N; Pettit, G R; Adsay, V; Vaitkevicius, V K; Al-Katib, A M; Sarkar, F H

    1999-11-01

    Adenocarcinoma of the pancreas generally remains an incurable disease by available treatment modalities, demanding the development of a suitable cell-culture/animal model and the discovery and evaluation of novel therapeutic agents. We report the clonal preservation of a human pancreatic cell line (KCI-MOH1) established from a 74-year-old African-American man diagnosed with pancreatic cancer. Initially the human primary tumor was grown as a xenograft in SCID mice and, subsequently, a cell line was established from tumors grown as a xenograft as reported in our earlier publication. The molecular characterization of the primary tumor, the tumors grown as xenograft, and the cell line all revealed similar genotypic properties. By using an automated DNA sequencer, a K-ras mutation (codon 12, GGT to CGT, Gly to Arg) was detected in the pancreatic tumor tissue taken from the patient, whereas no p53 mutation was detected. The same K-ras mutation and unaltered p53 was also found in the xenograft tumor and in the KCI-MOH1 cell line. Chromosome analysis of the cultured cells revealed: 42,XY,add(3)(p11.2),der(7)t(7;12) (p22;q12),-10,-12,add (14)(p11),-18,add (20)(q13),-22/84, idemx2, which is the same chromosome complement found in xenograft tumors. The KCI-MOH1 cell line grows well in tissue culture and forms tumors in the SCID mice when implanted subcutaneously, as well as in orthotopic sites. The KCI-MOH1 cell line-derived SCID mouse xenograft model was used for efficacy evaluation of bryostatin 1, auristatin-PE, spongistatin 1, and gemcitabine alone and in combination. Tumor growth inhibition (T/C expressed as percentage), tumor growth delay (T - C), and log 10 kill for these agents were 38%, 22 days, and 0.53; 15%, 30 days, and 0.80; 24%, 25 days, and 0.66; and 10%, 33 days, and 0.90, respectively. When given in combination, two of seven gemcitabine + auristatin-PE-treated animals were free of tumors for 150 days and were considered cured. Animals treated with a

  7. In vitro study of AFB1 and AFM1 effects on human lymphoblastoid Jurkat T-cell model.

    Science.gov (United States)

    Luongo, D; Russo, R; Balestrieri, A; Marzocco, S; Bergamo, P; Severino, L

    2014-10-01

    Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by Aspergillus spp. that can occur as a natural contaminant in foods and feeds of vegetable origin. Post-ingestion, AFB(1) can be metabolized in the liver of mammals into hydroxylated aflatoxin M(1) (AFM(1)) that is excreted with milk. Although several studies have been carried out to evaluate effects of AFB(1) on the immune system, studies regarding AFM(1) are moreover lacking. The aim of the current study was to investigate effects of AFB(1) and AFM(1) on immune function using a lymphoblastoid Jurkat T-cell line as an experimental model. Both AFB(1) and AFM(1) produced significant decreases in Jurkat cell proliferation, whereas only minor effects were noted on interleukin (IL)-2 and interferon (IFN)-γ cytokines mRNA expression in stimulated cells that had been pre-incubated with AFB(1) and AFM(1). Particularly, AFB(1), but not AFM(1), at the highest concentration (50 µM) induced a marked increase in IL-8 mRNA expression. The results of the current study suggested the existence of a concentration threshold for AFB(1) and AFM(1) needed to exert biological activity on cell viability and innate immunity.

  8. Multiple breast cancer cell-lines derived from a single tumor differ in their molecular characteristics and tumorigenic potential.

    Directory of Open Access Journals (Sweden)

    Goar Mosoyan

    Full Text Available BACKGROUND: Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient's breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT treatment. METHODS: Five breast cancer cell lines were derived from a single patient's primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER, CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC. In addition, a Fluorescent In Situ Hybridization (FISH assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. RESULTS: We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. CONCLUSIONS: All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to

  9. Growth of Coxiella burnetii in the Ixodes scapularis-derived IDE8 tick cell line.

    Science.gov (United States)

    Herrin, Brian; Mahapatra, Saugata; Blouin, Edmour F; Shaw, Edward I

    2011-07-01

    Q fever, a zoonotic disease, is caused by a gram-negative intracellular bacterium, Coxiella burnetii. Although normally transmitted during exposure to infectious aerosols, C. burnetii is also found in arthropod vectors. In the environment, ticks are thought to play a crucial role in bacterial maintenance and transmission by infecting various mammalian species. However, the nature of the pathogen-tick relationship is not well defined. To determine C. burnetii's interactions with a cultured tick cell line, we introduced purified C. burnetii NMII into Ixodes scapularis-derived IDE8 cells and assayed for bacterial presence, replication, gene expression, and subsequent infectivity for mammalian cells. Tick cells were harvested at 24 h, 72 h, 7 days, and 11 days postinfection (PI). C. burnetii uptake and subsequent replication was demonstrated by indirect immunofluorescence assay, electron microscopy, and real-time polymerase chain reaction (PCR). When a genome equivalent multiplicity of infection of 30 was used, 30%-40% of exposed cells were seen to have small, rounded, vacuoles at 72 h PI, whereas at 7 and 11 days PI, 60%-70% of cells contained enlarged vacuoles harboring large numbers of bacteria. Quantitative PCR analysis of total genomic DNA confirmed that C. burnetii genome numbers increased significantly from 24 h to 11 days PI. Expression of C. burnetii type four secretion system homologs at 7 days PI was demonstrated by reverse transcriptase PCR. Finally, indirect immunofluorescence assay demonstrated that C. burnetii propagated within IDE8 cells were infectious for mammalian cells. These studies demonstrate the utility of cultured tick cell lines as a model to investigate C. burnetii's molecular interactions with its arthropod vectors.

  10. MiRNA Profiles in Lymphoblastoid Cell Lines of Finnish Prostate Cancer Families.

    Directory of Open Access Journals (Sweden)

    Daniel Fischer

    Full Text Available Heritable factors are evidently involved in prostate cancer (PrCa carcinogenesis, but currently, genetic markers are not routinely used in screening or diagnostics of the disease. More precise information is needed for making treatment decisions to distinguish aggressive cases from indolent disease, for which heritable factors could be a useful tool. The genetic makeup of PrCa has only recently begun to be unravelled through large-scale genome-wide association studies (GWAS. The thus far identified Single Nucleotide Polymorphisms (SNPs explain, however, only a fraction of familial clustering. Moreover, the known risk SNPs are not associated with the clinical outcome of the disease, such as aggressive or metastasised disease, and therefore cannot be used to predict the prognosis. Annotating the SNPs with deep clinical data together with miRNA expression profiles can improve the understanding of the underlying mechanisms of different phenotypes of prostate cancer.In this study microRNA (miRNA profiles were studied as potential biomarkers to predict the disease outcome. The study subjects were from Finnish high risk prostate cancer families. To identify potential biomarkers we combined a novel non-parametrical test with an importance measure provided from a Random Forest classifier. This combination delivered a set of nine miRNAs that was able to separate cases from controls. The detected miRNA expression profiles could predict the development of the disease years before the actual PrCa diagnosis or detect the existence of other cancers in the studied individuals. Furthermore, using an expression Quantitative Trait Loci (eQTL analysis, regulatory SNPs for miRNA miR-483-3p that were also directly associated with PrCa were found.Based on our findings, we suggest that blood-based miRNA expression profiling can be used in the diagnosis and maybe even prognosis of the disease. In the future, miRNA profiling could possibly be used in targeted screening, together with Prostate Specific Antigene (PSA testing, to identify men with an elevated PrCa risk.

  11. Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation

    Directory of Open Access Journals (Sweden)

    Satish Kumar

    2016-01-01

    Full Text Available A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs.

  12. Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier.

    Science.gov (United States)

    Peng, Yu-Shiang; Lai, Po-Liang; Peng, Sydney; Wu, His-Chin; Yu, Siang; Tseng, Tsan-Yun; Wang, Li-Fang; Chu, I-Ming

    2014-01-01

    Parkinson's disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF) have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood-brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI) is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI) and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol) of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively) by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810). This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA) polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the polymer:DNA weight ratio of 10:1, which was 1.7-fold higher than the maximal GDNF expression of PEI1800-g-CHI/DNA. The low toxicity and high transfection efficiency of PEI600-g-CHI make it ideal for application to GDNF gene therapy, which has potential for the treatment of Parkinson's disease.

  13. 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...

  14. Comparison of human tenascin expression in normal, simian-virus-40-transformed and tumor-derived cell lines.

    Science.gov (United States)

    Carnemolla, B; Borsi, L; Bannikov, G; Troyanovsky, S; Zardi, L

    1992-04-15

    Tenascin is a polymorphic high-molecular-mass extracellular-matrix glycoprotein composed of six similar subunits. Using two-domain-specific anti-tenascin monoclonal antibodies, we have studied the expression and distribution of tenascin in four cultured normal human fibroblasts, two simian-virus-40-(SV40)-transformed and three tumor-derived (melanoma, rhabdomyosarcoma and fibrosarcoma) cell lines. We found that (a) cultured normal human fibroblasts accumulate considerable amounts of tenascin and retain 60-90% in the extracellular matrix, while they release the remainder into the tissue-culture medium; (b) of the two SV40-transformed counterparts we have tested, the AG-280 cell line accumulates no detectable amounts of tenascin and the WI-38-VA cell line accumulates about 10-times less tenascin than its normal counterpart and releases about 90% of it into the culture medium; (c) some tumor-derived cell lines accumulate considerable amounts of tenascin, but in these cases, more than 90% is released into the culture media; (d) in normal human fibroblasts, two major tenascin isoforms, generated by alternative splicing of the mRNA precursor, are detectable (280 kDa and 190 kDa, respectively) and the lower-molecular-mass tenascin isoform is accumulated preferentially in the extracellular matrix; (e) in SV40-transformed or tumor-derived cell lines, only the higher-molecular-mass isoform is detectable and it is more sialylated than the tenascin produced by the normal human fibroblast cell lines.

  15. Natural bizbenzoquinoline derivatives protect zebrafish lateral line sensory hair cells from aminoglycoside toxicity

    Directory of Open Access Journals (Sweden)

    Matthew eKruger

    2016-03-01

    Full Text Available Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20-30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment.

  16. The Non-Survival Effects of Glial Cell Line-Derived Neurotrophic Factor on Neural Cells

    Directory of Open Access Journals (Sweden)

    Daniel Cortés

    2017-08-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF was first characterized as a survival-promoting molecule for dopaminergic neurons (DANs. Afterwards, other cells were also discovered to respond to GDNF not only as a survival factor but also as a protein supporting other cellular functions, such as proliferation, differentiation, maturation, neurite outgrowth and other phenomena that have been less studied than survival and are now more extendedly described here in this review article. During development, GDNF favors the commitment of neural precursors towards dopaminergic, motor, enteric and adrenal neurons; in addition, it enhances the axonal growth of some of these neurons. GDNF also induces the acquisition of a dopaminergic phenotype by increasing the expression of Tyrosine Hydroxylase (TH, Nurr1 and other proteins that confer this identity and promote further dendritic and electrical maturation. In motor neurons (MNs, GDNF not only promotes proliferation and maturation but also participates in regenerating damaged axons and modulates the neuromuscular junction (NMJ at both presynaptic and postsynaptic levels. Moreover, GDNF modulates the rate of neuroblastoma (NB and glioblastoma cancer cell proliferation. Additionally, the presence or absence of GDNF has been correlated with conditions such as depression, pain, muscular soreness, etc. Although, the precise role of GDNF is unknown, it extends beyond a survival effect. The understanding of the complete range of properties of this trophic molecule will allow us to investigate its broad mechanisms of action to accelerate and/or improve therapies for the aforementioned pathological conditions.

  17. Axon guidance of sympathetic neurons to cardiomyocytes by glial cell line-derived neurotrophic factor (GDNF.

    Directory of Open Access Journals (Sweden)

    Keiko Miwa

    Full Text Available Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs and sympathetic neurons (SNs isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml or nerve growth factor (50 ng/ml. As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β1-adrenergic receptors (BAR with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.

  18. Axon guidance of sympathetic neurons to cardiomyocytes by glial cell line-derived neurotrophic factor (GDNF).

    Science.gov (United States)

    Miwa, Keiko; Lee, Jong-Kook; Takagishi, Yoshiko; Opthof, Tobias; Fu, Xianming; Hirabayashi, Masumi; Watabe, Kazuhiko; Jimbo, Yasuhiko; Kodama, Itsuo; Komuro, Issei

    2013-01-01

    Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of β1-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.

  19. Glial cell line-derived neurotrophic factor protects against high-fat diet-induced obesity.

    Science.gov (United States)

    Mwangi, Simon Musyoka; Nezami, Behtash Ghazi; Obukwelu, Blessing; Anitha, Mallappa; Marri, Smitha; Fu, Ping; Epperson, Monica F; Le, Ngoc-Anh; Shanmugam, Malathy; Sitaraman, Shanthi V; Tseng, Yu-Hua; Anania, Frank A; Srinivasan, Shanthi

    2014-03-01

    Obesity is a growing epidemic with limited effective treatments. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) was recently shown to enhance β-cell mass and improve glucose control in rodents. Its role in obesity is, however, not well characterized. In this study, we investigated the ability of GDNF to protect against high-fat diet (HFD)-induced obesity. GDNF transgenic (Tg) mice that overexpress GDNF under the control of the glial fibrillary acidic protein promoter and wild-type (WT) littermates were maintained on a HFD or regular rodent diet for 11 wk, and weight gain, energy expenditure, and insulin sensitivity were monitored. Differentiated mouse brown adipocytes and 3T3-L1 white adipocytes were used to study the effects of GDNF in vitro. Tg mice resisted the HFD-induced weight gain, insulin resistance, dyslipidemia, hyperleptinemia, and hepatic steatosis seen in WT mice despite similar food intake and activity levels. They exhibited significantly (PGDNF enhanced β-adrenergic-mediated cAMP release in brown adipocytes and suppressed lipid accumulation in differentiated 3T3L-1 cells through a p38MAPK signaling pathway. Our studies demonstrate a novel role for GDNF in the regulation of high-fat diet-induced obesity through increased energy expenditure. They show that GDNF and its receptor agonists may be potential targets for the treatment or prevention of obesity.

  20. Impairment of mineralization by metavanadate and decavanadate solutions in a fish bone-derived cell line.

    Science.gov (United States)

    Tiago, Daniel M; Laizé, Vincent; Cancela, M Leonor; Aureliano, Manuel

    2008-06-01

    Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short- and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 microM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85 +/- 0.40 versus 3.95 +/- 0.10 microg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 microM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems.

  1. Amino and nitro derivatives of 5,7-dimethoxyflavone from Kaempferia parviflora and cytotoxicity against KB cell line.

    Science.gov (United States)

    Wanich, Suchana; Yenjai, Chavi

    2009-09-01

    Structural modification of 5,7-dimethoxyflavone isolated from Kaempferia parviflora furnished two nitro and seven amino derivatives. Among these, six new (3, 5-6, 8-10) and three known (2, 4, 7) flavonoid derivatives were synthesized. All compounds were evaluated for cytotoxicity against KB cell line using colorimetric method. Compounds 6 and 8 exhibited strong cytotoxicity with IC50 values of 6.80 and 5.84 microg/mL, respectively.

  2. Transplantability of human lymphoid cell line, lymphoma, and leukemia in splenectomized and/or irradiated nude mice

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, S.; Shimosato, Y.; Kuroki, M.; Sato, Y.; Nakajima, T.

    1980-07-01

    The effects of splenectomy and/or whole-body irradiation of nude mice before xenotransplantation of lymphoid cell lines, lymphoma, and leukemia were studied. Transplantation after whole-body irradiation resulted in the increased ''take'' rate of three cultured cell lines (two of T-cell-derived acute lymphocytic leukemia and one of B-cell derived acute lymphocytic leukemia) and in the tumorous growth of Burkitt-derived Raji and spontaneously transformed lymphoblastoid cell lines. With splenectomy plus irradiation as a pretreatment, tumorous growth occurred in four other cell lines which were not transplantable after irradiation only (two cell lines of Epstein-Barr virus-transformed cord blood cells and one each of null acute lymphocytic leukemia and nodular lymphoma-derived cell lines). Direct transplantation of leukemia and lymphoma cells into the pretreated mice was successful in 7 of 24 cases (29%). B-cell-derived diffuse large lymphoid lymphoma was transplantable in three of seven cases (43%). However, lymphoma and leukemia of peripheral T-cell origin was difficult to transplant even with pretreatment, and only one pleomorphic T-cell lymphoma grew to a significant size (2 cm). One tumor each of B-cell-derived diffuse large lymphoid and T-cell diffuse lymphoblastic lymphoma became transplantable.

  3. Dysregulation of the TGF-β Postreceptor Signaling Pathway in Cell Lines Derived from Primary or Metastatic Ovarian Cancer

    Institute of Scientific and Technical Information of China (English)

    奚玲; 胡伟; 孟力; 周剑峰; 卢运萍; 王常玉; 马丁

    2004-01-01

    Summary: Transforming growth factor-beta (TGF-β) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF-β. Mechanisms of resistance to TGF-β caused by modulation of cell cycle regulators and/or inactivation of components of the TGF-β signaling transduction pathway such as C-myc and Smad4are not well understood. To investigate the potential association between loss of sensitivity to TGFβ and expression status of transforming growth factor receptor Ⅱ (TβR Ⅱ ), Smad4, CDC25A and C-myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi-quantitative RT-PCR. Normal ovarian surface tissues were used as controls. The expression of TβR Ⅱ was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88 % (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20 % (1/5) of non-tumorigenic cell lines (P<0.05). C-myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF-β of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF-β induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF-β is not associated with a lack of TβR Ⅱ.

  4. Glial cell line-derived neurotrophic factor gene delivery via a polyethylene imine grafted chitosan carrier

    Directory of Open Access Journals (Sweden)

    Peng YS

    2014-06-01

    Full Text Available Yu-Shiang Peng,1,* Po-Liang Lai,2,* Sydney Peng,1 His-Chin Wu,3 Siang Yu,1 Tsan-Yun Tseng,4 Li-Fang Wang,5 I-Ming Chu1 1Department of Chemical Engineering, National Tsing Hua University, Hsinchu, 2Department of Orthopedic Surgery, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, 3Department of Materials Engineering, Tatung University, Taipei, 4Graduate School of Biotechnology and Bioengineering, College of Engineering, Yuan Ze University, Chung-Li, 5Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan *Yu-Shiang Peng and Po-Liang Lai contributed equally to this work Abstract: Parkinson’s disease is known to result from the loss of dopaminergic neurons. Direct intracerebral injections of high doses of recombinant glial cell line-derived neurotrophic factor (GDNF have been shown to protect adult nigral dopaminergic neurons. Because GDNF does not cross the blood–brain barrier, intracerebral gene transfer is an ideal option. Chitosan (CHI is a naturally derived material that has been used for gene transfer. However, the low water solubility often leads to decreased transfection efficiency. Grafting of highly water-soluble polyethylene imines (PEI and polyethylene glycol onto polymers can increase their solubility. The purpose of this study was to design a non-viral gene carrier with improved water solubility as well as enhanced transfection efficiency for treating Parkinsonism. Two molecular weights (Mw =600 and 1,800 g/mol of PEI were grafted onto CHI (PEI600-g-CHI and PEI1800-g-CHI, respectively by opening the epoxide ring of ethylene glycol diglycidyl ether (EX-810. This modification resulted in a non-viral gene carrier with less cytotoxicity. The transfection efficiency of PEI600-g-CHI/deoxyribonucleic acid (DNA polyplexes was significantly higher than either PEI1800-g-CHI/DNA or CHI/DNA polyplexes. The maximal GDNF expression of PEI600-g-CHI/DNA was at the

  5. Polyketide Derivatives from Annona muricata Linn Leaves as Potencial Anticancer Material by Combination Treatment With Doxorubicin on Hela Cell Line

    Science.gov (United States)

    Artanti, A. N.; Astirin, O. P.; Prayito, A.; Widiyaningsih, R. F.; Prihapsara, F.

    2017-02-01

    One of the compounds found effication as an anticancer agent on cervical cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on hela cell line. An approach recently develop to overcome side effect of chemoterapeutic agent is used of combined chemoterapeutic agent, i.e doxorubicin. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). The expression of p53 profile was evaluated by immunohistochemistry on hela cell line. Data analysis showed that combination of polyketide derivative from Annona muricata L. (38,5 µg/ml) and doxorubicin with all of concentration performed synergistic effect on hela cell line with CI value from 0,33 – 0,65. The analysis on immucytochemistry showed that polyketide derivative from Annona muricata L. leaves could enhance p53 pathway significantly on hela cell line.

  6. The procurement of cells for the derivation of human embryonic stem cell lines for therapeutic use: recommendations for good practice.

    Science.gov (United States)

    Murdoch, Alison; Braude, Peter; Courtney, Aidan; Brison, Daniel; Hunt, Charles; Lawford-Davies, James; Moore, Harry; Stacey, Glyn; Sethe, Sebastian

    2012-03-01

    The donation of human embryos for the derivation of embryonic stem cell lines that may be used in the development of therapeutic products raises more complex ethical, practical and regulatory problems than the donation of embryos for non-clinical research. This review considers these issues and offers recommendations for good practice.

  7. Association between smoking behaviour and genetic variants of glial cell line-derived neurotrophic factor

    Indian Academy of Sciences (India)

    ESZTER KOTYUK; NORA NEMETH; ZSOLT RONAI; ZSOLT DEMETROVICS; MARIA SASVARI-SZEKELY; ANNA SZEKELY

    2016-12-01

    Glial cell line-derived neurotrophic factor (GDNF) promotes development and differentiation of dopaminergic neurons, thus it has an important role in dopamine-related neuropsychiatric disorders. Since the role of dopamine system in smoking iswell established, we hypothesized that GDNF gene variants may affect smoking behaviour. Self-reported data on smoking behaviour (never smoked, quit, occasional, or regular smokers) and level of nicotine addiction (Hooked on Nicotine Checklist and Fagerstrom Nicotine Addiction Scale), anxiety, as well as buccal samples were obtained from 930 Hungarian young adults (18–35 years). Genetic analysis involved eight GDNF single-nucleotide polymorphisms (SNP) (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111). Allele-wise association analyses of the eight GDNF SNPs provided a significant association between smoking behaviour and rs3096140 (P = 0.0039). The minor allele (C) was more frequent in those groups who smoked in some form (quit, occasional or regular smokers) as compared to those who neversmoked (P = 0.0046). This result remained significant after Bonferroni correction for multiple testing. In the ever smoking group, no significant differences were found in the level of nicotine addiction by the alleles of these polymorphisms. Also, nosignificant interaction of rs3096140 and smoking categories were observed on anxiety mean scores. Although previous data demonstrated an association between GDNF rs2910704 and severity of methamphetamine use to the best of our knowledge, this is the first study on the role of GDNF genetic variations in smoking behaviour. Our results suggest that GDNF rs3096140 might be involved in the genetic background of smoking, independent of anxiety characteristics.

  8. Dopamine receptor activation increases glial cell line-derived neurotrophic factor in experimental stroke.

    Science.gov (United States)

    Kuric, Enida; Wieloch, Tadeusz; Ruscher, Karsten

    2013-09-01

    Treatment with levodopa enhances functional recovery after experimental stroke but its mechanisms of action are elusive. Reactive astrocytes in the ischemic hemisphere are involved in mechanisms promoting recovery and also express dopamine 1 (D1) and dopamine 2 (D2) receptors. Here we investigated if the activation of astrocytic dopamine receptors (D1 and D2) regulates the expression of glial cell line-derived neurotrophic factor (GDNF) after combined in vitro hypoxia/aglycemia (H/A) and studied the expression of GDNF in the ischemic brain after treatment with levodopa/benserazide following transient occlusion of the middle cerebral artery (tMCAO) in the rat. Twenty-four hours after H/A, GDNF levels were upregulated in exposed astrocytes compared to normoxic control cultures and further elevated by the addition of the selective D1 receptor agonist (R)-(+)-SKF-38393 hydrochloride while D1 receptor antagonism by R(+)-SCH-23390 hydrochloride significantly reduced GDNF. No effect on GDNF levels was observed by the application of the D2 receptor agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide hydrate or S-(-)-eticlopride hydrochloride (D2 receptor antagonist). After tMCAO, GDNF was upregulated in D1 expressing reactive astrocytes in the peri-infarct area. In addition, treatment with levodopa/benserazide significantly increased GDNF levels in the infarct core and peri-infarct area after tMCAO without affecting the expression of glial fibrillar acidic protein (GFAP), an intermediate filament and marker of reactive gliosis. After stroke, GDNF levels increase in the ischemic hemisphere in rats treated with levodopa, implicating GDNF in the mechanisms of tissue reorganization and plasticity and in l-DOPA enhanced recovery of lost brain function. Our results support levodopa treatment as a potential recovery enhancing therapy in stroke patients.

  9. Glial cell line-derived neurotrophic factor induces cell proliferation in the mouse urogenital sinus.

    Science.gov (United States)

    Park, Hyun-Jung; Bolton, Eric C

    2015-02-01

    Glial cell line-derived neurotrophic factor (GDNF) is a TGFβ family member, and GDNF signals through a glycosyl-phosphatidylinositol-linked cell surface receptor (GFRα1) and RET receptor tyrosine kinase. GDNF signaling plays crucial roles in urogenital processes, ranging from cell fate decisions in germline progenitors to ureteric bud outgrowth and renal branching morphogenesis. Gene ablation studies in mice have revealed essential roles for GDNF signaling in urogenital development, although its role in prostate development is unclear. We investigated the functional role of GDNF signaling in the urogenital sinus (UGS) and the developing prostate of mice. GDNF, GFRα1, and RET show time-specific and cell-specific expression during prostate development in vivo. In the UGS, GDNF and GFRα1 are expressed in the urethral mesenchyme (UrM) and epithelium (UrE), whereas RET is restricted to the UrM. In each lobe of the developing prostate, GDNF and GFRα1 expression declines in the epithelium and becomes restricted to the stroma. Using a well-established organ culture system, we determined that exogenous GDNF increases proliferation of UrM and UrE cells, altering UGS morphology. With regard to mechanism, GDNF signaling in the UrM increased RET expression and phosphorylation of ERK1/2. Furthermore, inhibition of RET kinase activity or ERK kinases suppressed GDNF-induced proliferation of UrM cells but not UrE cells. We therefore propose that GDNF signaling in the UGS increases proliferation of UrM and UrE cells by different mechanisms, which are distinguished by the role of RET receptor tyrosine kinase and ERK kinase signaling, thus implicating GDNF signaling in prostate development and growth.

  10. Presynaptic modulation of spinal nociceptive transmission by glial cell line-derived neurotrophic factor (GDNF).

    Science.gov (United States)

    Salio, Chiara; Ferrini, Francesco; Muthuraju, Sangu; Merighi, Adalberto

    2014-10-01

    The role of glial cell line-derived neurotrophic factor (GDNF) in nociceptive pathways is still controversial, as both pronociceptive and antinociceptive actions have been reported. To elucidate this role in the mouse, we performed combined structural and functional studies in vivo and in acute spinal cord slices where C-fiber activation was mimicked by capsaicin challenge. Nociceptors and their terminals in superficial dorsal horn (SDH; laminae I-II) constitute two separate subpopulations: the peptidergic CGRP/somatostatin+ cells expressing GDNF and the nonpeptidergic IB4+ neurons expressing the GFRα1-RET GDNF receptor complex. Ultrastructurally the dorsal part of inner lamina II (LIIid) harbors a mix of glomeruli that either display GDNF/somatostatin (GIb)-IR or GFRα1/IB4 labeling (GIa). LIIid thus represents the preferential site for ligand-receptor interactions. Functionally, endogenous GDNF released from peptidergic CGRP/somatostatin+ nociceptors upon capsaicin stimulation exert a tonic inhibitory control on the glutamate excitatory drive of SDH neurons as measured after ERK1/2 phosphorylation assay. Real-time Ca(2+) imaging and patch-clamp experiments with bath-applied GDNF (100 nM) confirm the presynaptic inhibition of SDH neurons after stimulation of capsaicin-sensitive, nociceptive primary afferent fibers. Accordingly, the reduction of the capsaicin-evoked [Ca(2+)]i rise and of the frequency of mEPSCs in SDH neurons is specifically abolished after enzymatic ablation of GFRα1. Therefore, GDNF released from peptidergic CGRP/somatostatin+ nociceptors acutely depresses neuronal transmission in SDH signaling to nonpeptidergic IB4+ nociceptors at glomeruli in LIIid. These observations are of potential pharmacological interest as they highlight a novel modality of cross talk between nociceptors that may be relevant for discrimination of pain modalities.

  11. Association between smoking behaviour and genetic variants of glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Kotyuk, Eszter; Nemeth, Nora; Ronai, Zsolt; Demetrovics, Zsolt; Sasvari-Szekely, Maria; Szekely, Anna

    2016-12-01

    Glial cell line-derived neurotrophic factor (GDNF) promotes development and differentiation of dopaminergic neurons, thus it has an important role in dopamine-related neuropsychiatric disorders. Since the role of dopamine system in smoking is well established, we hypothesized that GDNF gene variants may affect smoking behaviour. Self-reported data on smoking behaviour (never smoked, quit, occasional, or regular smokers) and level of nicotine addiction (Hooked on Nicotine Checklist and Fagerstrom Nicotine Addiction Scale), anxiety, as well as buccal samples were obtained from 930 Hungarian young adults (18-35 years). Genetic analysis involved eight GDNF single-nucleotide polymorphisms (SNP) (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111). Allele-wise association analyses of the eight GDNF SNPs provided a significant association between smoking behaviour and rs3096140 (P=0.0039). The minor allele (C) was more frequent in those groups who smoked in some form (quit, occasional or regular smokers) as compared to those who never smoked (P = 0.0046). This result remained significant after Bonferroni correction for multiple testing. In the ever smoking group, no significant differences were found in the level of nicotine addiction by the alleles of these polymorphisms. Also, no significant interaction of rs3096140 and smoking categories were observed on anxiety mean scores. Although previous data demonstrated an association between GDNF rs2910704 and severity of methamphetamine use to the best of our knowledge, this is the first study on the role of GDNF genetic variations in smoking behaviour. Our results suggest that GDNF rs3096140 might be involved in the genetic background of smoking, independent of anxiety characteristics.

  12. Glial cell line-derived neurotrophic factor (GDNF) as a novel candidate gene of anxiety.

    Science.gov (United States)

    Kotyuk, Eszter; Keszler, Gergely; Nemeth, Nora; Ronai, Zsolt; Sasvari-Szekely, Maria; Szekely, Anna

    2013-01-01

    Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for dopaminergic neurons with promising therapeutic potential in Parkinson's disease. A few association analyses between GDNF gene polymorphisms and psychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder and drug abuse have also been published but little is known about any effects of these polymorphisms on mood characteristics such as anxiety and depression. Here we present an association study between eight (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111) GDNF single nucleotide polymorphisms (SNPs) and anxiety and depression scores measured by the Hospital Anxiety and Depression Scale (HADS) on 708 Caucasian young adults with no psychiatric history. Results of the allele-wise single marker association analyses provided significant effects of two single nucleotide polymorphisms on anxiety scores following the Bonferroni correction for multiple testing (p = 0.00070 and p = 0.00138 for rs3812047 and rs3096140, respectively), while no such result was obtained on depression scores. Haplotype analysis confirmed the role of these SNPs; mean anxiety scores raised according to the number of risk alleles present in the haplotypes (p = 0.00029). A significant sex-gene interaction was also observed since the effect of the rs3812047 A allele as a risk factor of anxiety was more pronounced in males. In conclusion, this is the first demonstration of a significant association between the GDNF gene and mood characteristics demonstrated by the association of two SNPs of the GDNF gene (rs3812047 and rs3096140) and individual variability of anxiety using self-report data from a non-clinical sample.

  13. Glial cell line-derived neurotrophic factor (GDNF as a novel candidate gene of anxiety.

    Directory of Open Access Journals (Sweden)

    Eszter Kotyuk

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF is a neurotrophic factor for dopaminergic neurons with promising therapeutic potential in Parkinson's disease. A few association analyses between GDNF gene polymorphisms and psychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder and drug abuse have also been published but little is known about any effects of these polymorphisms on mood characteristics such as anxiety and depression. Here we present an association study between eight (rs1981844, rs3812047, rs3096140, rs2973041, rs2910702, rs1549250, rs2973050 and rs11111 GDNF single nucleotide polymorphisms (SNPs and anxiety and depression scores measured by the Hospital Anxiety and Depression Scale (HADS on 708 Caucasian young adults with no psychiatric history. Results of the allele-wise single marker association analyses provided significant effects of two single nucleotide polymorphisms on anxiety scores following the Bonferroni correction for multiple testing (p = 0.00070 and p = 0.00138 for rs3812047 and rs3096140, respectively, while no such result was obtained on depression scores. Haplotype analysis confirmed the role of these SNPs; mean anxiety scores raised according to the number of risk alleles present in the haplotypes (p = 0.00029. A significant sex-gene interaction was also observed since the effect of the rs3812047 A allele as a risk factor of anxiety was more pronounced in males. In conclusion, this is the first demonstration of a significant association between the GDNF gene and mood characteristics demonstrated by the association of two SNPs of the GDNF gene (rs3812047 and rs3096140 and individual variability of anxiety using self-report data from a non-clinical sample.

  14. Incorporation study of {sup 18}FDG through its uptake into tumour-derived cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Costa, Flavia M.; Nascimento, Leonardo T.C.; Santos, Raquel G., E-mail: fnc@cdtn.br, E-mail: raqueou@uol.com.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN-CNEN/MG), Belo Horizonte, MG (Brazil)

    2013-07-01

    In vitro tests of radiopharmaceuticals are an important instrument to study their mechanisms of action, binding and incorporation. {sup 18}FDG is the most used radiopharmaceutical for diagnostics positron emission tomography (PET) on oncology, on the basis of accelerated rates of absorption of glucose in cell malignancies. This radiotracer has been routinely produced at CDTN; and therefore, it was selected for preliminary assays due to its availability. Nowadays, UPPR at CDTN produces routinely {sup 18}FDG for the local PET Centers but others PET radiopharmaceuticals are in development such as {sup 18}F-Fluorocholine and {sup 18}F-Fluorothymidine. According to the Brazilian Health Regulatory Agency (ANVISA) it is necessary to validate and register these new radiopharmaceuticals in order to get the approval for their commercialization. Targeting efficacy is one of the important issues to be evaluated during radiopharmaceutical validation. The aim of this study was to develop a standard protocol to determine tumor targeting efficacy of PET radiopharmaceuticals in vitro. Therefore it was developed a protocol based on the incorporation og {sup 18} FDG through the uptake in different tumor-derived cell lines. Three variables were investigated for the standardization of the test: the number of cells to be seeded in 96-well plates, the time of incubation with the radiopharmaceutical and the radiotracer concentration. The standardized protocol was considered suitable for {sup 18}FDG incorporation assay and showed reproductive results. The protocol developed in this work will pave the way for the in vitro studies of incorporation of the new PET radiopharmaceuticals to be produced at UPPR-CDTN, such as: {sup 18}F-Fluorocholine and {sup 18}F-Fluorothymidine. (author)

  15. MIO-M1 cells and similar muller glial cell lines derived from adult human retina exhibit neural stem cell characteristics

    National Research Council Canada - National Science Library

    Lawrence, Jean M; Singhal, Shweta; Bhatia, Bhairavi; Keegan, David J; Reh, Thomas A; Luthert, Philip J; Khaw, Peng T; Limb, Gloria Astrid

    2007-01-01

    .... We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO-M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors...

  16. LINES

    Directory of Open Access Journals (Sweden)

    Minas Bakalchev

    2015-10-01

    Full Text Available The perception of elements in a system often creates their interdependence, interconditionality, and suppression. The lines from a basic geometrical element have become the model of a reductive world based on isolation according to certain criteria such as function, structure, and social organization. Their traces are experienced in the contemporary world as fragments or ruins of a system of domination of an assumed hierarchical unity. How can one release oneself from such dependence or determinism? How can the lines become less “systematic” and forms more autonomous, and less reductive? How is a form released from modernistic determinism on the new controversial ground? How can these elements or forms of representation become forms of action in the present complex world? In this paper, the meaning of lines through the ideas of Le Corbusier, Leonidov, Picasso, and Hitchcock is presented. Spatial research was made through a series of examples arising from the projects of the architectural studio “Residential Transformations”, which was a backbone for mapping the possibilities ranging from playfulness to exactness, as tactics of transformation in the different contexts of the contemporary world.

  17. Echinococcus granulosus-specific T-cell lines derived from patients at various clinical stages of cystic echinococcosis.

    Science.gov (United States)

    Riganò, R; Buttari, B; De Falco, E; Profumo, E; Ortona, E; Margutti, P; Scottà, C; Teggi, A; Siracusano, A

    2004-01-01

    To investigate the role of T lymphocytes in the immune response to Echinococcus granulosus, using sheep hydatid fluid (SHF) and antigen B (AgB), we generated T-cell lines from patients with active, transitional and inactive hydatid cysts. We established 16 T-cell lines, eight specific to SHF and eight specific to AgB. At surface phenotyping 88-98% of cells displayed the helper/inducer CD4 antigen. In all patients, at all clinical stages of hydatid cyst disease, T-cell stimulation with SHF and AgB invariably amplified a large number of almost identical Vbeta subfamily fragments. Irrespective of antigen-specificity, the two cell lines from the patient with an inactive cyst had a Th1 profile, because they exclusively expressed and produced IFN-gamma. Conversely, the T-cell lines derived from the seven patients with active and transitional hydatid cysts had mixed Th1/Th2 and Th0 clones. The functional characteristics of the 16 T-cell lines differed markedly in the various clinical stages of cystic echinococcosis, thus providing new in vitro evidence that Th1 lymphocytes contribute decisively to the inactive stage of hydatid disease, Th2 lymphocytes in the active and transitional stages. The parasite-specific T-cell lines, especially the two Th1 lines from the patient with an inactive cyst, may help identify Th1 protective epitopes on SHF and AgB.

  18. Hepatic progenitor cell lines from allyl alcohol-treated adult rats are derived from gamma-irradiated mouse STO cells.

    Science.gov (United States)

    Zhang, Mingjun; Sell, Stewart; Leffert, Hyam L

    2003-01-01

    In attempts to recharacterize several markers of putative rat liver progenitor cells, single-stage reverse transcription-polymerase chain reaction (RT-PCR) analyses failed to confirm the reported immunochemical detection of albumin, alpha(1)-fetoprotein, and cytochrome P450-1A2 in the clonal line, 3(8)#21, and the cloned derivative, 3(8)#21-EGFP (enhanced green fluorescent protein). Undetectable expression occurred whether or not both lines were cultured on or off feeder layers of gamma-irradiated mouse embryonic STO (SIM [Sandoz inbred Swiss mouse] thioguanine-resistant ouabain-resistant) cells. PCR amplification of liver progenitor cell chromosomal (rat and mouse Pigr, rat INS1, mouse INS2) and mitochondrial (rat and mouse COX1) genes revealed only mouse sequences. Further analyses of rat and mouse COX1 sequences in cells from untampered storage vials of all 11 reported liver progenitor cell lines and strains revealed only mouse sequences. In addition, uniquely similar metaphase spreads were observed in STO, 3(8)#21, and 3(8)#21-EGFP cells. The combined results suggest that the previously reported "rat" liver progenitor cell lines were most likely generated during early derivation in cell culture from gamma-radiation-resistant or ineffectively irradiated mouse STO cells used as the feeder layers. These findings reveal new types of artifacts encountered in cocultures of tissue progenitor cells and feeder layer cell lines, and they sound a cautionary note: phenotypic and genotypic properties of feeder layers should be well-characterized before and during coculture with newly derived stem cells and clonal derivatives.

  19. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    Science.gov (United States)

    Hammer, Susanne C.; Becker, Annegret; Rateitschak, Katja; Mohr, Annika; Lüder Ripoli, Florenza; Hennecke, Silvia; Junginger, Johannes; Hewicker-Trautwein, Marion; Brenig, Bertram; Ngezahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

    2016-01-01

    Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies. PMID:27690019

  20. Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells.

    Science.gov (United States)

    Santner, S J; Dawson, P J; Tait, L; Soule, H D; Eliason, J; Mohamed, A N; Wolman, S R; Heppner, G H; Miller, F R

    2001-01-01

    The MCF10 series of cell lines was derived from benign breast tissue from a woman with fibrocystic disease. The MCF10 human breast epithelial model system consists of mortal MCF10M and MCF10MS (mortal cells grown in serum-free and serum-containing media, respectively), immortalized but otherwise normal MCF10F and MCF10A lines (free-floating versus growth as attached cells), transformed MCF10AneoT cells transfected with T24 Ha-ras, and premalignant MCF10AT cells with potential for neoplastic progression. The MCF10AT, derived from xenograft-passaged MCF10-AneoT cells, generates carcinomas in approximately 25% of xenografts. We now report the derivation of fully malignant MCF10CA1 lines that complete the spectrum of progression from relatively normal breast epithelial cells to breast cancer cells capable of metastasis. MCF10CA1 lines display histologic variations ranging from undifferentiated carcinomas, sometimes with focal squamous differentiation, to well-differentiated adenocarcinomas. At least two metastasize to the lung following injection of cells into the tail vein; one line grows very rapidly in the lung, with animals moribund within 4 weeks, whereas the other requires 15 weeks to reach the same endpoint. In addition to variations in efficiency of tumor production, the MCF10CA1 lines show differences in morphology in culture, anchorage-independent growth, karyotype, and immunocytochemistry profiles. The MCF10 model provides a unique tool for the investigation of molecular changes during progression of human breast neoplasia and the generation of tumor heterogeneity on a common genetic background.

  1. Buttressing staples with cholecyst-derived extracellular matrix (CEM) reinforces staple lines in an ex vivo peristaltic inflation model.

    LENUS (Irish Health Repository)

    Burugapalli, Krishna

    2008-11-01

    Staple line leakage and bleeding are the most common problems associated with the use of surgical staplers for gastrointestinal resection and anastomotic procedures. These complications can be reduced by reinforcing the staple lines with buttressing materials. The current study reports the potential use of cholecyst-derived extracellular matrix (CEM) in non-crosslinked (NCEM) and crosslinked (XCEM) forms, and compares their mechanical performance with clinically available buttress materials [small intestinal submucosa (SIS) and bovine pericardium (BP)] in an ex vivo small intestine model.

  2. Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

    Science.gov (United States)

    Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

    2011-06-01

    The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

  3. Cytotoxic, Antiproliferative and Apoptotic Effects of New Benzimidazole Derivatives on A549 Lung Carcinoma and C6 Glioma Cell Lines.

    Science.gov (United States)

    Yurttas, Leyla; Demirayak, Seref; Ciftci, Gulsen Akalın

    2015-01-01

    Benzimidazole ring is a versatile structure which has been extensively utilized in medicinal chemistry. Since we are working on 1,2-disubstutited benzimidazoles, we have reported new antitumor active derivatives. As a continuation to our previous work, we have synthesized a new series of 1-(2-aryl-2-oxoethyl)-2-[(N,Ndimethylamino/pyrrolidinyl/piperidinyl)thiocarbamoyl] benzimidazole derivatives. Anticancer activity of the compounds was evaluated using MTT assay, BrdU assay and flow cytometric analysis on A549 human lung carcinoma and C6 rat glioma cell lines. Compounds bearing dimethylamino moiety exhibited higher antitumor activity.

  4. Genetic instability of cell lines derived from a single human small cell carcinoma of the lung

    DEFF Research Database (Denmark)

    Engelholm, S A; Vindeløv, L L; Spang-Thomsen, M

    1985-01-01

    with different DNA content appeared. By cloning, permanent cell lines were established from the new subpopulations, whereas the original population stopped growing. The cloned cell lines were characterized by morphology, chromosomes analysis, electron microscopy and plating efficiency; the stability of the DNA......Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations...

  5. Physical Parameters of a Flare Derived from Multi-line 2D Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Time series of 2D spectra of Hα and CaII λ8542 for a flare of 1999 December 22 are obtained and analyzed with a new fitting technique. The method we proposed can simultaneously yield the four parameters: the line source function, the optical thickness at line center, the line-of-sight velocity and the Doppler width. We present the spatial distributions of the physical parameters and their temporal evolutions determined from the 2D spectra. Our results are consistent with the general picture predicted by the flare dynamic models.

  6. The niche-derived glial cell line-derived neurotrophic factor (GDNF induces migration of mouse spermatogonial stem/progenitor cells.

    Directory of Open Access Journals (Sweden)

    Lisa Dovere

    Full Text Available In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF, a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

  7. The niche-derived glial cell line-derived neurotrophic factor (GDNF) induces migration of mouse spermatogonial stem/progenitor cells.

    Science.gov (United States)

    Dovere, Lisa; Fera, Stefania; Grasso, Margherita; Lamberti, Dante; Gargioli, Cesare; Muciaccia, Barbara; Lustri, Anna Maria; Stefanini, Mario; Vicini, Elena

    2013-01-01

    In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

  8. A One Line Derivation of DCC: Application of a Vector Random Coefficient Moving Average Process

    NARCIS (Netherlands)

    C.M. Hafner (Christian); M.J. McAleer (Michael)

    2014-01-01

    markdownabstract__Abstract__ One of the most widely-used multivariate conditional volatility models is the dynamic conditional correlation (or DCC) specification. However, the underlying stochastic process to derive DCC has not yet been established, which has made problematic the derivation of asym

  9. Cell Lines Derived From Feline Fibrosarcoma Display Unstable Chromosomal Aneuploidy and Additionally Centrosome Number Aberrations

    National Research Council Canada - National Science Library

    Erichsen, J. von; Hecht, W; Löhberg-Gruene, C; Reinacher, M

    2012-01-01

    The purpose of the study was to evaluate clonality and presence of numerical chromosomal and centrosomal aberrations in 5 established feline fibrosarcoma cell lines and in a fetal dermal fibroblast...

  10. Synthesis of 2,3-diyne-1,4-naphthoquinone derivatives and evaluation of cytotoxic activity against tumor cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Mauro G.; Camara, Celso A.; Silva, Tania M.S., E-mail: ccelso@dcm.ufrpe.br [Universidade Federal Rural de Pernambuco (LSCB/UFRPE), Recife, PE (Brazil). Dept. de Ciencias Moleculares. Lab. de Sintese de Compostos Bioativos; Feitosa, Anderson C.S.; Meira, Assuero S.; Pessoa, Claudia [Universidade Federal do Ceara (LOE/UFC), Fortaleza, CE (Brazil). Dept. de Fisiologia e Farmacologia. Lab. de Oncologia Experimental

    2013-09-15

    A series of 2,3-diyne-1,4-naphthoquinone derivatives was synthesized from 2,3-dibromo- 1,4-naphthoquinone and various functionalized terminal alkynes using palladium-catalyzed Sonogashira cross-coupling reaction. The diynes were evaluated as potential cytotoxic agents against three tumor cell lines: human ovarian adenocarcinoma (OVCAR-8), human metastatic prostate cancer (PC-3M) and human bronchoalveolar lung carcinoma (NCI-H358M), presenting, in general, satisfactory results for inhibition of cell growth. (author)

  11. Image Prediction Method with Nonlinear Control Lines Derived from Kriging Method with Extracted Feature Points Based on Morphing

    Directory of Open Access Journals (Sweden)

    Kohei Arai

    2013-01-01

    Full Text Available Method for image prediction with nonlinear control lines which are derived from extracted feature points from the previously acquired imagery data based on Kriging method and morphing method is proposed. Through comparisons between the proposed method and the conventional linear interpolation and widely used Cubic Spline interpolation methods, it is found that the proposed method is superior to the conventional methods in terms of prediction accuracy.

  12. The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking.

    Science.gov (United States)

    Englund, Mikael C O; Caisander, Gunilla; Noaksson, Karin; Emanuelsson, Katarina; Lundin, Kersti; Bergh, Christina; Hansson, Charles; Semb, Henrik; Strehl, Raimund; Hyllner, Johan

    2010-04-01

    This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.

  13. Diverse HLA-I Peptide Repertoires of the APC Lines MUTZ3-Derived Immature and Mature Dendritic Cells and THP1-Derived Macrophages.

    Science.gov (United States)

    Nyambura, Lydon Wainaina; Jarmalavicius, Saulius; Baleeiro, Renato Brito; Walden, Peter

    2016-09-15

    Dendritic cells (DCs) and macrophages are specialized APCs that process and present self-Ags for induction of tolerance and foreign Ags to initiate T cell-mediated immunity. Related to differentiation states they have specific phenotypes and functions. However, the impact of these differentiations on Ag processing and presentation remains poorly defined. To gain insight into this, we analyzed and compared the HLA-I peptidomes of MUTZ3-derived human immature and mature DC lines and THP1-derived macrophages by liquid chromatography tandem mass spectrometry. We found that the HLA-I peptidomes were heterogeneous and individualized and were dominated by nonapeptides with similar HLA-I binding affinities and anchor residues. MUTZ3-derived DCs and THP1-derived macrophages were able to sample peptides from source proteins of almost all subcellular locations and were involved in various cellular functions in similar proportion, with preference to proteins involved in cell communication, signal transduction, protein metabolism, and transcription factor/regulator activity.

  14. Aperture corrections for disk galaxy properties derived from the CALIFA survey. Balmer emission lines in spiral galaxies

    CERN Document Server

    Iglesias-Páramo, J; Galbany, L; Sánchez, S F; Rosales-Ortega, F F; Mast, D; García-Benito, R; Husemann, B; Aguerri, J A L; Alves, J; Bekeraité, S; Bland-Hawthorn, J; Catalán-Torrecilla, C; de Amorim, A L; de Lorenzo-Cáceres, A; Ellis, S; Falcón-Barroso, J; Flores, H; Florido, E; Gallazzi, A; Gomes, J M; Delgado, R M González; Haines, T; Hernández-Fernández, J D; Kehrig, C; López-Sánchez, A R; Lyubenova, M; Marino, R A; Mollá, M; Monreal-Ibero, A; Mourão, A; Papaderos, P; Sánchez-Blázquez, P; Spekkens, K; Stanishev, V; van de Ven, G; Walcher, C J; Wisotzki, L; Zibetti, S; Ziegler, B

    2013-01-01

    This work investigates the effect of the aperture size on derived galaxy properties for which we have spatially-resolved optical spectra. We focus on some indicators of star formation activity and dust attenuation for spiral galaxies that have been widely used in previous work on galaxy evolution. We have used 104 spiral galaxies from the CALIFA survey for which 2D spectroscopy with complete spatial coverage is available. From the 3D cubes we have derived growth curves of the most conspicuous Balmer emission lines (Halpha, Hbeta) for circular apertures of different radii centered at the galaxy's nucleus after removing the underlying stellar continuum. We find that the Halpha flux (f(Halpha)) growth curve follows a well defined sequence with aperture radius showing low dispersion around the median value. From this analysis, we derive aperture corrections for galaxies in different magnitude and redshift intervals. Once stellar absorption is properly accounted for, the f(Halpha)/f(Hbeta) ratio growth curve shows...

  15. Establishment and Characterization of Fibroblast Cell Line Derived from Siberian Tiger (Panthera tigris altaica).

    Science.gov (United States)

    Liu, Changqing; Guo, Yu; Liu, Dan; Guan, Weijun; Ma, Yuehui

    2010-06-01

    The Siberian tiger ear marginal tissue fibroblast cell line (STF34) from 34 samples was successfully established using primary explants technique and cell cryoconservation technology. STF34 cells were adherent, with a population doubling time of 24 h. Chromosome analysis showed that 90.2%-91.6% of cells were diploid (2n = 38). Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that STF34 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses, and mycoplasmas were all negative. Every index of the STF34 cell line meets all the standard quality controls of American Type Culture Collection. Not only has the germline of this important Siberian tiger species been preserved at the cell level, but also valuable material had been provided for genome, postgenome, and somacloning research.

  16. A cell line derived from the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae).

    Science.gov (United States)

    Goodman, Cynthia L; Stanley, David; Ringbauer, Joseph A; Beeman, Richard W; Silver, Kristopher; Park, Yoonseong

    2012-08-01

    The red flour beetle, Tribolium castaneum, is a model organism for agricultural and medical research and its complete genome is sequenced. We established a continuously replicating T. castaneum cell line to complement existing physiological, genetic, and genomic research tools. We set up trial cell cultures from egg, pupa, and adult stages as tissue sources and incubated them in six separate cell culture media to determine the optimal combination of tissue source and medium for cell replication. Our most promising culture was generated by co-culturing adult (∼75 %) and pupal tissues in EX-CELL 420 medium containing 9 % FBS. Our new cell culture is designated BCIRL-TcA-CLG1 (TcA) and it has been subcultured more than 90 times. Amplification of genomic DNA with species-specific primers yielded DNA fragments of the expected sizes and with sequences identical to those from the published Tribolium genome. Additionally, we characterized this line using DNA fingerprinting (DAF-PCR) and compared it with three other coleopteran cell lines and its conspecific pupae to confirm identity. Its doubling time is 155.2 hr. Early passages consisted of attached cells and vesicles in suspension, whereas later passages consisted primarily of attached, spherical cells. Similar to other established cell lines, the ploidy of TcA cells was variable, ranging from 20 chromosomes/cell (diploid) to above 30 chromosomes/cell. TcA cells withstood incubation at 40°C for 1 h with no decrease in viability. We recorded increased levels of one heat shock protein (43 kDa) and of the hsp68a transcript following exposure to 40°C. Taken together, this represents the first report of a continuously replicating T. castaneum cell line. We expect the BCIRL-TcA-CLG1 line will become a useful tool in Tribolium research.

  17. Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

    DEFF Research Database (Denmark)

    Feuerborn, Alexander; Moritz, Constanze; von Bonin, Frederike;

    2006-01-01

    Classical Hodgkin's lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the pathology of cHL, the transforming events remain to be elucidated. It has been proposed that mutations in the TP53 gene in biopsy material as well as cell lines ...... loss of exons 10 - 11 (L1236) or exons 8 - 11 (HDLM-2), respectively. These changes were found in otherwise rarely mutated regions of TP53. Cell lines L1236 and HDLM-2 harbour fusions with alu-repeats in their TP53 mRNA 3'-ends, resulting in the carboxyterminal truncation and loss...

  18. Characterisation of a mouse tumour cell line with in vitro derived resistance to verapamil.

    OpenAIRE

    Twentyman, P. R.; Wright, K A; Fox, N. E.

    1990-01-01

    We have established a subline (EMT6/VRP) of the mouse tumour cell line EMT6/P with acquired resistance to the calcium transport blocker verapamil (VRP). The subline was 4-fold resistant to the cytoxicity of VRP alone compared with the parent line but of similar sensitivity to adriamycin, vincristine or colchicine. EMT6/VRP cells growing in 75 micrograms ml-1 VRP were morphologically different from and larger in diameter than EMT6/P cells, but these two parameters reverted almost to normal wit...

  19. Characterisation of a mouse tumour cell line with in vitro derived resistance to verapamil.

    OpenAIRE

    Twentyman, P. R.; Wright, K A; Fox, N. E.

    1990-01-01

    We have established a subline (EMT6/VRP) of the mouse tumour cell line EMT6/P with acquired resistance to the calcium transport blocker verapamil (VRP). The subline was 4-fold resistant to the cytoxicity of VRP alone compared with the parent line but of similar sensitivity to adriamycin, vincristine or colchicine. EMT6/VRP cells growing in 75 micrograms ml-1 VRP were morphologically different from and larger in diameter than EMT6/P cells, but these two parameters reverted almost to normal wit...

  20. Immunophenotypic, cytogenetic, and mutational characterization of cell lines derived from myelodysplastic syndrome patients after progression to acute myeloid leukemia.

    Science.gov (United States)

    Palau, Anna; Mallo, Mar; Palomo, Laura; Rodríguez-Hernández, Ines; Diesch, Jeannine; Campos, Diana; Granada, Isabel; Juncà, Jordi; Drexler, Hans G; Solé, Francesc; Buschbeck, Marcus

    2017-03-01

    Leukemia cell lines have been widely used in the hematology field to unravel mechanistic insights and to test new therapeutic strategies. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of diseases that are characterized by ineffective hematopoiesis and frequent progress to acute myeloid leukemia (AML). A few cell lines have been established from MDS patients after progression to AML but their characterization is incomplete. Here we provide a detailed description of the immunophenotypic profile of the MDS-derived cell lines SKK-1, SKM-1, F-36P; and MOLM-13. Specifically, we analyzed a comprehensive panel of markers that are currently applied in the diagnostic routine for myeloid disorders. To provide high-resolution genetic data comprising copy number alterations and losses of heterozygosity we performed whole genome single nucleotide polymorphism-based arrays and included the cell line OHN-GM that harbors the frequent chromosome arm 5q deletion. Furthermore, we assessed the mutational status of 83 disease-relevant genes. Our results provide a resource to the MDS and AML field that allows researchers to choose the best-matching cell line for their functional studies. © 2016 Wiley Periodicals, Inc.

  1. Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

    Institute of Scientific and Technical Information of China (English)

    WANG Juan; GAO Ya-nan; KONG Yue-qin; JIANG Jin-jin; LI Ai-min; ZHANG Yong-tai; WANG You-ping

    2014-01-01

    Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The lforal organ of the sterile line SaNa-1A was sterile with a shortened iflament and delfated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

  2. Optimization of protocols for derivation of mouse embryonic stem cell lines from refractory strains, including the non obese diabetic mouse.

    Science.gov (United States)

    Davies, Timothy J; Fairchild, Paul J

    2012-07-01

    The derivation of pluripotent embryonic stem cells (ESCs) from a variety of genetic backgrounds remains a desirable objective in the generation of mice functionally deficient in genes of interest and the modeling of human disease. Nevertheless, disparity in the ease with which different strains of mice yield ESC lines has long been acknowledged. Indeed, the generation of bona fide ESCs from the non obese diabetic (NOD) mouse, a well-characterized model of human type I diabetes, has historically proved especially difficult to achieve. Here, we report the development of protocols for the derivation of novel ESC lines from C57Bl/6 mice based on the combined use of high concentrations of leukemia inhibitory factor and serum-replacement, which is equally applicable to fresh and cryo-preserved embryos. Further, we demonstrate the success of this approach using Balb/K and CBA/Ca mice, widely considered to be refractory strains. CBA/Ca ESCs contributed to the somatic germ layers of chimeras and displayed a very high competence at germline transmission. Importantly, we were able to use the same protocol for the derivation of ESC lines from nonpermissive NOD mice. These ESCs displayed a normal karyotype that was robustly stable during long-term culture, were capable of forming teratomas in vivo and germline competent chimeras after injection into recipient blastocysts. Further, these novel ESC lines efficiently formed embryoid bodies in vitro and could be directed in their differentiation along the dendritic cell lineage, thus illustrating their potential application to the generation of cell types of relevance to the pathogenesis of type I diabetes.

  3. Characterization of cell lines derived from breast cancers and normal mammary tissues for the study of the intrinsic molecular subtypes.

    Science.gov (United States)

    Prat, Aleix; Karginova, Olga; Parker, Joel S; Fan, Cheng; He, Xiaping; Bixby, Lisa; Harrell, J Chuck; Roman, Erick; Adamo, Barbara; Troester, Melissa; Perou, Charles M

    2013-11-01

    Five molecular subtypes (luminal A, luminal B, HER2-enriched, basal-like, and claudin-low) with clinical implications exist in breast cancer. Here, we evaluated the molecular and phenotypic relationships of (1) a large in vitro panel of human breast cancer cell lines (BCCLs), human mammary fibroblasts (HMFs), and human mammary epithelial cells (HMECs); (2) in vivo breast tumors; (3) normal breast cell subpopulations; (4) human embryonic stem cells (hESCs); and (5) bone marrow-derived mesenchymal stem cells (hMSC). First, by integrating genomic data of 337 breast tumor samples with 93 cell lines we were able to identify all the intrinsic tumor subtypes in the cell lines, except for luminal A. Secondly, we observed that the cell lines recapitulate the differentiation hierarchy detected in the normal mammary gland, with claudin-low BCCLs and HMFs cells showing a stromal phenotype, HMECs showing a mammary stem cell/bipotent progenitor phenotype, basal-like cells showing a luminal progenitor phenotype, and luminal B cell lines showing a mature luminal phenotype. Thirdly, we identified basal-like and highly migratory claudin-low subpopulations of cells within a subset of triple-negative BCCLs (SUM149PT, HCC1143, and HCC38). Interestingly, both subpopulations within SUM149PT were enriched for tumor-initiating cells, but the basal-like subpopulation grew tumors faster than the claudin-low subpopulation. Finally, claudin-low BCCLs resembled the phenotype of hMSCs, whereas hESCs cells showed an epithelial phenotype without basal or luminal differentiation. The results presented here help to improve our understanding of the wide range of breast cancer cell line models through the appropriate pairing of cell lines with relevant in vivo tumor and normal cell counterparts.

  4. Quality characteristics of soybean pasted (Doenjang) manufactured with 2 soybean mutant lines derived from cv. baekwon

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kyung Jun; Kang, Si Yong; Choi, Hong Il; Kim, Jin Baek [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2016-11-15

    In order to identification of the possibility of manufacturing soybean paste (doenjang) with soybean mutant lines induced from gamma-ray mutagenesis, this study was performed to investigate the quality characteristics of doenjang using two soybean mutant lines, Baekwon-1 (BW-1) and Baekwon-2 (BW-2) and their original cultivar (cv. Baekwon, BW) for 8 weeks. The BW and two mutant lines (BW-1 and BW-2) were showed higher content of amino type nitrogen than control (cv. Taegwang). The pH decreased and the titratable acidity increased all the samples during aging period. The lightness, redness and yellowness of doenjang were the lowest in BW. Total free sugar content of doenjang was the highest in control (10.43%) after 4 weeks and composed mainly fructose and glucose. The order of the free amino acid content was Glutamic acid>Leucine>Lysine>Phenylalanine>Aspartic acid in control, Glutamic acid>Leucine >Arginine>Lysine>Phenylalanine in BW, Glutamic acid>Lysine>Phenylalanine>Aspartic acid>Valine in BW-1 and Glutamic acid>Arginine>Lysine>Phenylalanine>Aspartic acid in BW-2, respectively. Our results showed that it is possible to increase the quality of doenjang using soybean mutant lines in manufacturing soybean paste.

  5. Density diagnostics derived from the OIV and SIV intercombination lines observed by IRIS

    CERN Document Server

    Polito, V; Dudík, J; Mason, H E; Giunta, A; Reeves, K K

    2016-01-01

    The intensity of the \\oiv~2s$^{2}$ 2p $^{2}$P-2s2p$^{2}$ $^{4}$P and \\siv~3 s$^{2}$ 3p $^{2}$P- 3s 3p$^{2}$ $^{4}$ P intercombination lines around 1400~\\AA~observed with the \\textit{Interface Region Imaging Spectrograph} (IRIS) provide a useful tool to diagnose the electron number density ($N_\\textrm{e}$) in the solar transition region plasma. We measure the electron number density in a variety of solar features observed by IRIS, including an active region (AR) loop, plage and brightening, and the ribbon of the 22-June-2015 M 6.5 class flare. By using the emissivity ratios of \\oiv\\ and \\siv\\ lines, we find that our observations are consistent with the emitting plasma being near isothermal (log$T$[K] $\\approx$ 5) and iso-density ($N_\\textrm{e}$ $\\approx$~10$^{10.6}$ cm$^{-3}$) in the AR loop. Moreover, high electron number densities ($N_\\textrm{e}$ $\\approx$~10$^{13}$ cm$^{-3}$) are obtained during the impulsive phase of the flare by using the \\siv\\ line ratio. We note that the \\siv\\ lines provide a higher ran...

  6. Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark

    DEFF Research Database (Denmark)

    Harkness, Linda; Rasmussen, Iben Anne; Erb, Karin

    2010-01-01

    . Analysis of clinical data showed that the majority of embryos (94.5%) failed to reach the blastocyst stage of development and of all embryos, regardless of developmental status, 248 embryos were needed to create one stem cell line. From the number of embryos (69) which developed to the blastocyst stage 8...

  7. Fate of Aegilops speltoides-derived, repetitive DNA sequences in diploid Aegilops species, wheat-Aegilops amphiploids and derived chromosome addition lines.

    Science.gov (United States)

    Kumar, S; Friebe, B; Gill, B S

    2010-07-01

    The present study reports the cloning and characterization of an Aegilops speltoides-derived subtelomeric repeat, designated as pSp1B16. Clone pSp1B16 has 98% sequence homology with the previously isolated Ae. speltoides repeat Spelt1. The distribution of pSp1B16 and another Ae. speltoides repeat, pGc1R1, was analyzed in diploid Aegilops species, tetra- and hexaploid wheats, wheat-Aegilops amphiploids and derived chromosome addition lines by fluorescence in situ hybridization (FISH). Clones pSp1B16 and pGc1R1 revealed FISH sites in Ae. speltoides, Ae. sharonensis and Triticum timopheevii, whereas additional pGc1R1 FISH sites were observed in Ae. longissima and Ae. caudata. The pSp1B16 and pGc1R1 FISH patterns of the Aegilops chromosomes in the wheat-Aegilops amphiploids and chromosome addition lines are similar to those present in the Aegilops parent accession. We did not observe any evidence of pSp1B16 and pGc1R1 sequence elimination, which is in contrast to previous studies using similar hybrids and repeats. The presented data suggest that the genomic changes in synthetic amphiploids observed in previous studies might be caused by homoeologous recombination, which was suppressed in the amphiploid analyzed in this study.

  8. Density diagnostics derived from the O iv and S iv intercombination lines observed by IRIS

    Science.gov (United States)

    Polito, V.; Del Zanna, G.; Dudík, J.; Mason, H. E.; Giunta, A.; Reeves, K. K.

    2016-10-01

    The intensity of the O iv 2s2 2p 2P-2s2p24P and S iv 3 s2 3p 2P-3s 3p24 P intercombination lines around 1400 Å observed with the Interface Region Imaging Spectrograph (IRIS) provide a useful tool to diagnose the electron number density (Ne) in the solar transition region plasma. We measure the electron number density in a variety of solar features observed by IRIS, including an active region (AR) loop, plage and brightening, and the ribbon of the 22-June-2015 M 6.5 class flare. By using the emissivity ratios of O iv and S iv lines, we find that our observations are consistent with the emitting plasma being near isothermal (logT[K] ≈ 5) and iso-density (Ne ≈ 1010.6 cm-3) in the AR loop. Moreover, high electron number densities (Ne ≈ 1013 cm-3) are obtained during the impulsive phase of the flare by using the S iv line ratio. We note that the S iv lines provide a higher range of density sensitivity than the O iv lines. Finally, we investigate the effects of high densities (Ne ≳ 1011 cm-3) on the ionization balance. In particular, the fractional ion abundances are found to be shifted towards lower temperatures for high densities compared to the low density case. We also explored the effects of a non-Maxwellian electron distribution on our diagnostic method. The movie associated to Fig. 3 is available at http://www.aanda.org

  9. A cell line resource derived from honey bee (Apis mellifera embryonic tissues.

    Directory of Open Access Journals (Sweden)

    Michael J Goblirsch

    Full Text Available A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz's L15 medium and incubated at 32(°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711 has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32 and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1 gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10-14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.

  10. A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

    Science.gov (United States)

    Goblirsch, Michael J.; Spivak, Marla S.; Kurtti, Timothy J.

    2013-01-01

    A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology. PMID:23894551

  11. Characterization of murine pituitary-derived cell lines Tpit/F1, Tpit/E and TtT/GF.

    Science.gov (United States)

    Yoshida, Saishu; Higuchi, Masashi; Ueharu, Hiroki; Nishimura, Naoto; Tsuda, Mitsuyoshi; Yako, Hideji; Chen, Mo; Mitsuishi, Hideo; Sano, Yoshiya; Kato, Takako; Kato, Yukio

    2014-01-01

    The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100β and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.

  12. Dose-dependent cytotoxic and mutagenic effects of antineoplastic alkylating agents on human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Sanderson, B.J.S.; Johnson, K.J.; Henner, W.D. (Oregon Health Sciences Univ., Portland (United States))

    1991-01-01

    The alkylating agents in clinical use as antineoplastics are strongly implicated as human carcinogens on the basis of animal studies and human epidemiologic studies. However, there is little quantitative information on the extent to which exposure to these drugs is mutagenic for normal (non-malignant) cells and the extent to which such mutagenicity correlates with cytotoxicity of these agents. Human lymphoblastoid cells (WIL2-NS) were exposed to graded doses of eight antineoplastic alkylating agents. Dose-dependent decreases in survival were used to calculate IC{sub 50}s for each of the drugs tested. The mutagenicity of these agents is correlated strongly with cytotoxicity. These results quantitate the dose-dependent cytotoxic and mutagenic effects of these bifunctional alkylating agents on human cells. All are cytotoxic and mutagenic, although their mutagenic efficiency varies.

  13. JTorX: A Tool for On-Line Model-Driven Test Derivation and Execution

    NARCIS (Netherlands)

    Belinfante, Axel; Esparza, Javier; Majumdar, Rupak

    We introduce JTorX, a tool for model-driven test derivation and execution, based on the ioco theory. This theory, originally presented in [Tretmans,1996], has been refined in [Tretmans,2008] with test-cases that are input-enabled. For models with underspecified traces [vdBijl+,2004] introduced

  14. Trustworthy Variant Derivation with Translation Validation for Safety Critical Product Lines

    DEFF Research Database (Denmark)

    Iosif-Lazăr, Alexandru Florin; Wasowski, Andrzej

    2016-01-01

    Software product line (SPL) engineering facilitates development of entire families of software products with systematic reuse. Model driven SPLs use models in the design and development process. In the safety critical domain, validation of models and testing of code increases the quality of the p......Software product line (SPL) engineering facilitates development of entire families of software products with systematic reuse. Model driven SPLs use models in the design and development process. In the safety critical domain, validation of models and testing of code increases the quality...... translation validation. We demonstrate it using Featherweight VML—a core language for separate variability modeling relying on a single kind of variation point to define transformations of artifacts seen as object models. We use Featherweight VML with its semantics as a correctness specification...

  15. Differential effects and transport kinetics of ascorbate derivatives in leukemic cell lines.

    Science.gov (United States)

    Koh, W S; Lee, S J; Lee, H; Park, C; Park, M H; Kim, W S; Yoon, S S; Park, K; Hong, S I; Chung, M H; Park, C H

    1998-01-01

    In order to investigate the differential effects of ascorbate derivatives on leukemic cell growth, we examined their stabilities and transmembrane transport efficiencies. The growth of HL-60 and U937 cells was dose-dependently inhibited by ascorbic acid and sodium ascorbate, but not by dehydroascorbic acid and magnesium ascorbyl 2-phosphate up to 200 microM. The growth-suppression by ascorbic acid was dependent on its redox state, showing a complete or partial reversion by ascorbate oxidase or FeCl3 addition, respectively. Three different patterns of intracellular ascorbic acid accumulation were observed by HPLC according to the species of ascorbate derivative applied for the incubation. Compared with the reduced form of ascorbic acid, the oxidized forms (dehydroascorbic acid, ascorbic acid plus ascorbate oxidase or FeCl3) were rapidly transported into cells and readily degraded, while magnesium ascorbyl 2-phosphate, a stable derivative of ascorbic acid, slowly elevated the intracellular level of ascorbic acid, reaching a plateau at 24 hours. We also measured the differential kinetics of ascorbic acid levels In culture supernatants following the addition of ascorbate derivatives. Ascorbic acid at 40, 10, or 1 microM was observed 3 hours following treatment with 100 microM of ascorbic acid, ascorbic acid plus FeCl3, or magnesium ascorbyl 2-phosphate, respectively. No ascorbic acid was found in the culture supernatant treated with dehydroascorbic acid. This order of ascorbic acid concentrations in culture supernatant reflects their growth-inhibitory effects. Thus the growth inhibitory effect of ascorbic acid appears to be dependent on its concentration in culture medium rather than its intracellular concentration. In conclusion, the results in this study indicate that the differential effects of ascorbate derivatives appear to be due to the actual concentration differences of the reduced form of ascorbic acid in culture medium following their addition, which is

  16. Derivation of Color Confusion Lines for Pseudo-Dichromat Observers from Color Discrimination Thresholds

    Directory of Open Access Journals (Sweden)

    Kahiro Matsudaira

    2011-05-01

    Full Text Available The objective is to develop a method of defining color confusion lines in the display RGB color space through color discrimination tasks. In the experiment, reference and test square patches were presented side by side on a CRT display. The subject's task is to set the test color where the color difference from the reference is just noticeable to him/her. In a single trial, the test color was only adjustable along one of 26 directions around the reference. Thus 26 colors with just noticeable difference (JND were obtained and made up a tube-like or an ellipsoidal shape around each reference. With color-anomalous subjects, the major axes of these shapes should be parallel to color confusion lines that have a common orientation vector corresponding to one of the cone excitation axes L, M, or S. In our method, the orientation vector was determined by minimizing the sum of the squares of the distances from JND colors to each confusion line. To assess the performance the method, the orientation vectors obtained by pseudo-dichromats (color normal observers with a dichromat simulator were compared to those theoretically calculated from the color vision model used in the simulator.

  17. A small-world network derived from the deterministic uniform recursive tree by line graph operation

    Science.gov (United States)

    Hou, Pengfeng; Zhao, Haixing; Mao, Yaping; Wang, Zhao

    2016-03-01

    The deterministic uniform recursive tree ({DURT}) is one of the deterministic versions of the uniform recursive tree ({URT}). Zhang et al (2008 Eur. Phys. J. B 63 507-13) studied the properties of DURT, including its topological characteristics and spectral properties. Although DURT shows a logarithmic scaling with the size of the network, DURT is not a small-world network since its clustering coefficient is zero. Lu et al (2012 Physica A 391 87-92) proposed a deterministic small-world network by adding some edges with a simple rule in each DURT iteration. In this paper, we intoduce a method for constructing a new deterministic small-world network by the line graph operation in each DURT iteration. The line graph operation brings about cliques at each node of the previous given graph, and the resulting line graph possesses larger clustering coefficients. On the other hand, this operation can decrease the diameter at almost one, then giving the analytic solutions to several topological characteristics of the model proposed. Supported by The Ministry of Science and Technology 973 project (No. 2010C B334708); National Science Foundation of China (Nos. 61164005, 11161037, 11101232, 11461054, 11551001); The Ministry of education scholars and innovation team support plan of Yangtze River (No. IRT1068); Qinghai Province Nature Science Foundation Project (Nos. 2012-Z-943, 2014-ZJ-907).

  18. Phenotypic and molecular characterization of selected tomato recombinant inbred lines derived from the cross Solanum lycopersicum × S. pimpinellifolium

    Indian Academy of Sciences (India)

    Guillermo R. Pratta; Gustavo R. Rodriguez; Roxana Zorzoli; Estela M. Valle; Liliana A. Picardi

    2011-08-01

    An important trait defining fresh tomato marketability is fruit shelf life. Exotic germplasm of Solanum pimpinellifolium is able to prolong shelf life. Sixteen recombinant inbred lines with differing values of shelf life and fruit weight were derived by antagonistic-divergent selection from an interspecific cross involving Solanum pimpinellifolium. The objective of this study was to evaluate these recombinant inbred lines for many fruit quality traits such as diameter, height, size, acidity, colour, firmness, shelf life and weight, and to characterize them by amplified fragment length polymorphism markers. For most traits, a wide range of genetic variability was found and a wide range of molecular variation was also detected. Both sets of data allowed the identification of recombinant inbred lines by means of cluster analysis and principal component analysis. Genetic association among some amplified fragment length polymorphism markers and fruit quality traits, suggested by the principal component analysis, could be identified by single point analysis. Potential molecular markers underlying agronomical traits were detected in these recombinant inbred lines.

  19. Variations in Humanized and Defined Culture Conditions Supporting Derivation of New Human Embryonic Stem Cell Lines

    DEFF Research Database (Denmark)

    Fletcher, Judy M; Ferrier, Patricia M; Gardner, John O

    2006-01-01

    and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products......, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission....

  20. Synthesis and apoptotic activity of new pyrazole derivatives in cancer cell lines.

    Science.gov (United States)

    Nitulescu, George Mihai; Draghici, Constantin; Olaru, Octavian Tudorel; Matei, Lilia; Ioana, Aldea; Dragu, Laura Denisa; Bleotu, Coralia

    2015-09-01

    We designed and synthesized new pyrazole thiourea chimeric derivatives and confirmed their structures by NMR and IR spectra. Apoptotic effects were studied in human cancer cells. The N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea compound (4b) exhibited the highest apoptosis-inducing effect. Compound 4b and the thiazole derivatives, 5b and 6b, increased the expression of tumor necrosis factor receptors TRAIL-R2 and TRAIL-R1, accompanied by down-modulation of pro-caspase 3 levels, and the augmentation of cleaved caspase 3. They also reduced the levels of apoptosis inhibitory proteins and the expression of the heat-shock proteins Hsp27 and Hsp70. All the tested pyrazole derivatives induced a concentration-dependent increase of cells in G2/M phases. The analysis of the experimental data indicates the reduction of Akt phosphorylation as the most probable cellular mechanism of action for the tested compounds. The in vitro study indicated that compound 4b could be a promising anti-cancer drug, to be further developed in animal models of cancer.

  1. Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow.

    Directory of Open Access Journals (Sweden)

    Atsushi Nagai

    Full Text Available Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs and mesenchymal stem cells (MSCs. MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10, was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1, neurons (neurofilament protein, synapsin and MAP2, astrocytes (glial fibrillary acidic protein, GFAP and oligodendrocytes (myelin basic protein, MBP as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells, neurofilament protein and beta-tubulin III (neurons GFAP (astrocytes, and galactocerebroside (oligodendrocytes. Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.

  2. Properties of an EBV-B cell line derived interleukin 1 (IL 1) receptor

    Energy Technology Data Exchange (ETDEWEB)

    Matsushima, K.; Akahoshi, T.; Yamada, M.; Furutani, Y.; Oppenheim, J.J.

    1986-03-01

    The properties of an human IL 1 receptor on a human EBV-B line were studied. Purified human IL 1-..beta.. produced by a myelomonocytic cell line (THP-1) was labeled with /sup 125/I by the Bolton-Hunter method without loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the most /sup 125/I IL-..beta... Maximal binding was reached within 20 min at 4/sup 0/C. Scatchard analysis of the binding of /sup 125/I-IL 1-..beta.. to VDS-O cells yielded a Kd of 2.4-5.9 x 10/sup -00/ M with 110 to 220 binding (receptor) sites/cell. The binding of /sup 125/I-IL 1-..beta.. to VDS-O cells was inhibited by anti-human IL 1 antibody, natural and recombinant human IL 1-..cap alpha.. as well as IL 1-..beta.., but not by IFN-..cap alpha.., TNF, or LT, suggesting that IL 1-..cap alpha.. and IL 1-..beta.. specifically bind to the same receptor. The mw of the IL 1 receptor on human EBV-B cells was estimated to be 60 Kd both by a chemical crosslinking method and by HPLC gel filtration analysis of solubilized receptor extracted from membranes by a nonionic detergent (CHAPS). The pI of solubilized human IL 1 receptor was 7.3 by HPLC chromatofocusing. Data showing that VDS-O cells proliferate in response to exogenously added IL 1, express IL 1 receptors and also produce IL 1 all support the hypothesis that IL 1 may function as an autocrine signal for B lymphocytes.

  3. Ins1 Gene Up-Regulated in a β-Cell Line Derived from Ins2 Knockout Mice

    OpenAIRE

    2003-01-01

    The authors have derived a new β-cell line (βIns2−/−lacZ) from Ins2−/− mice that carry the lacZ reporter gene under control of the Ins2 promoter. βIns2−/−lacZ cells stained positively using anti-insulin antibody, expressed β-cell–specific genes encoding the transcription factor PDX-1, glucokinase, and Glut-2, retained glucose-responsiveness for insulin secretion, and expressed the lacZ gene. Analysis of Ins1 expression by reverse transcriptase–polymerase chain reaction (RT-PCR) showed that In...

  4. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas

    2002-01-01

    -mediated transfer of the gene for human glial cell line-derived neurotrophic factor (GDNF) to embryonic (E27/28) porcine VM tissue kept as organotypic explant cultures. Treatment of the developing VM with two mitogens, basic fibroblast growth factor and epidermal growth factor, prior to transfection significantly...... numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo...

  5. Methoxyflavone derivatives modulate the effect of TRAIL-induced apoptosis in human leukemic cell lines

    Directory of Open Access Journals (Sweden)

    Wudtiwai Benjawan

    2011-12-01

    Full Text Available Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL induces apoptosis in various tumor cells, but does not affect normal cells or human leukemic cells, such as MOLT-4 and U937 cells, which are relatively resistant to TRAIL. Three flavonoids extracted from the rhizome of K. parviflora were 5,7-dimethoxyflavone (DMF, 5,7,4'-trimethoxyflavone (TMF and 3,5,7,3',4'-pentamethoxyflavone (PMF, and synthetic flavonoids including 5-methoxyflavone (5-MF and 2'-methoxyflavone (2"-MF were chosen for testing in this study. The aims of this study were to examine whether the treatment of TRAIL-resistant leukemia MOLT-4 and U937 cells, with methoxyflavone derivatives could enhance the apoptotic response and to identify the mechanism involved. Methods The cytotoxic effect of methoxyflavone (MF derivatives in MOLT-4, U937 and peripheral blood mononuclear cells (PBMCs was analyzed by the MTT assay. The induction of apoptosis and the reduction of mitochondrial transmembrane potential (ΔΨm after staining with annexin V FITC and propidium iodide (PI, and 3,3'-dihexyloxacarbocyanine iodide (DiOC6, respectively, were performed using flow cytometry. ROS production was determined by staining with 2',7'-dichlorofluorescin diacetate and processed with a flow cytometer. DR4, DR5, cFLIP, Mcl-1, BAX and Bid expression were demonstrated by immunoblotting. Caspase-8 and -3 activities were determined by using IETD-AFC and DEVD-AFC substrates and the fluorescence intensity was measured. Results All methoxyflavone derivatives were cytotoxic to MOLT-4, U937 cells and PBMCs, except DMF, TMF and PMF were not toxic to PBMCs. All MF derivatives induced human leukemic MOLT-4 cell apoptosis, but not in U937 cells. Percentage of MOLT-4 cells with (ΔΨm was increased when treated with DMF, TMF, PMF, 5-MF and 2'-MF in the presence of TRAIL. 5-MF and 2'-MF enhanced TRAIL-induced apoptosis through the up-regulation of both DRs and the down-regulation of c

  6. Establishment and characterization of immortal hepatocytes derived from various transgenic mouse lines.

    Science.gov (United States)

    Klocke, Rainer; Gómez-Lechón, M José; Ehrhardt, Anja; Mendoza-Figueroa, Tomas; Donato, M Teresa; López-Revilla, Rubén; Castell, José V; Paul, Dieter

    2002-06-21

    The potential of three genetic changes introduced into mice by the transgenic or knockout technology aimed at immortalizing hepatocytes in vitro and concomitantly preserving their differentiated hepatic functions was analyzed. Six hepatocyte lines were isolated from neonatal and adult transgenic mice expressing either IgEGF (a secretable variant of hEGF) or SV40 T antigen in the liver and from neonatal and adult p53 knockout (KO) mice and have been subcultured >150 times in serum-free, arginine-deficient medium. Only in SV40 T antigen transgenic lines profiles of mRNAs encoding serum proteins, transcription factors, and liver-specific enzymes were similar to those found in livers and primary hepatocytes. Accordingly, these cells displayed basal and inducible expression of CYP proteins as well as testosterone metabolizing activities. Thus, either knockout of the p53 gene or expression of SV40 T antigen or of IgEGF imparts immortality to hepatocytes in vitro, but only SV40 T antigen expression is compatible with the concomitant long-term preservation of differentiated liver functions.

  7. Establishment and cryopreservation of a fibroblast cell line derived from Bengal tiger (Panthera tigris tigris).

    Science.gov (United States)

    Guan, W J; Liu, C Q; Li, C Y; Liu, D; Zhang, W X; Ma, Y H

    2010-01-01

    The Bengal tiger ear marginal tissue fibroblasts cell line (BTF22), containing 157 tubes of frozen cells, was successfully established by using primary explants technique and cell cryoconservation technology. Biological analysis showed that the population doubling time (PDT) for revival cells was approximately 28 h. Measurement of LDH and MDH isoenzymes showed no cross-contamination among the cells. Karyotyping showed that the frequency of cells with chromosome number 2n = 38 was 90.6-92.2%. Tests for bacteria, fungi, viruses and mycoplasma were negative. Plasmids encoding the fluorescent proteins pEGFP-N3, pEGFP-C1, pECFP-N1, pECFP-mito, pDsRed1-N1, and pEYFP-N1 were transfected into cells to study exogenous gene expression in the cells. The plasmid transfection efficiency was between 4.4% and 31.9%. Every index of the BTF22 cell line meets all the standard quality controls of American type Culture Collection (ATCC). Not only has the germline of this important Bengal tiger species been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform would provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.

  8. OBLIQUE DERIVATIVE PROBLEMS FOR SECOND ORDER NONLINEAR MIXED EQUATIONS WITH DEGENERATE LINE

    Institute of Scientific and Technical Information of China (English)

    Wen Guochun

    2008-01-01

    The present article deals with oblique derivative problems for some nonlin-ear mixed equations with parabolic degeneracy, which include the Tricomi problem as a special case. First, the formulation of the problems for the equations is given; next, the representation and estimates of solutions for the above problems are obtained; finally, the existence of solutions for the problems is proved by the successive iteration and the com-pactness principle of solutions of the problems. In this article, the author uses the complex method, namely, the complex functions in the elliptic domain and the hyperbolic complex functions in hyperbolic domain are used.

  9. Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena).

    Science.gov (United States)

    Kim, Min Sung; Lee, Seung Tae; Lim, Jeong Mook; Gong, Seung Pyo

    2016-01-01

    This study was conducted to identify optimal medium composition for freezing Oryzias dancena embryonic cell lines. Different freezing media consisting of various concentration of dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and trehalose were prepared and long-term cultured embryonic cell line was frozen in each freezing medium by conventional slow freezing program for 7 days. Through measurement of viability and growth of post-thaw cells frozen in each freezing medium, it was determined that optimal composition of three components was 10 % DMSO, 20 % FBS, and 0.1 M trehalose. The post-thaw cells frozen in optimal freezing medium showed similar morphology and growth rate with non-frozen cells. Next, this condition was applied to two different sets of experiment; (1) freezing of the same cells during expanded period (57 days) and (2) freezing of short-term cultured cells from other batches for 7 days. The viability of post-thaw cells was significantly low and comparable in set 1 and 2, respectively, when compared with the result of long term-cultured cells frozen in optimal freezing medium for 7 days and similar morphology and growth rate with non-frozen counterparts were detected in the post-thaw cells from both sets. In conclusion, this study first reports the optimal medium composition for freezing O. dancena embryonic cells, which can contribute to fish species preservation as well as improvement of cell-based biotechnology by providing stable cell storage.

  10. An Abel transform for deriving line-of-sight wind profiles from LEO-LEO infrared laser occultation measurements

    Science.gov (United States)

    Syndergaard, S.; Kirchengast, G.

    2016-03-01

    We have developed a formula for the retrieval of the line-of-sight (l.o.s.) wind speed from future low Earth orbit (LEO) satellite-to-satellite infrared laser occultation measurements. The formula involves an Abelian integral transform akin to the Abel transform widely used for deriving refractive index from bending angle in Global Navigation Satellite System radio occultation measurements. Besides the Abelian integral transform, the formula is derived from a truncated series expansion of the volume absorption coefficient as a function of frequency and includes a simple absorption-line-asymmetry correction term. A first-order formulation (referred to as the standard formula) is complemented by higher-order terms that can be used for high-accuracy computations. Under the assumptions of spherical symmetry and perfect knowledge of spectroscopy, the residual l.o.s. wind error from using the standard formula rather than the high-accuracy formula is assessed to be small compared to that anticipated from measurement errors in a real experiment. Applying the new formula just in standard form to future infrared laser transmission profiles would therefore enable the retrieval of l.o.s. stratospheric wind profiles with an accuracy limited mainly by measurement errors, residual spectroscopic errors, and deviations from spherical symmetry.

  11. Inhibition of Cancer Derived Cell Lines Proliferation by Synthesized Hydroxylated Stilbenes and New Ferrocenyl-Stilbene Analogs. Comparison with Resveratrol

    Directory of Open Access Journals (Sweden)

    Malik Chalal

    2014-06-01

    Full Text Available Further advances in understanding the mechanism of action of resveratrol and its application require new analogs to identify the structural determinants for the cell proliferation inhibition potency. Therefore, we synthesized new trans-resveratrol derivatives by using the Wittig and Heck methods, thus modifying the hydroxylation and methoxylation patterns of the parent molecule. Moreover, we also synthesized new ferrocenylstilbene analogs by using an original protective group in the Wittig procedure. By performing cell proliferation assays we observed that the resveratrol derivatives show inhibition on the human colorectal tumor SW480 cell line. On the other hand, cell viability/cytotoxicity assays showed a weaker effects on the human hepatoblastoma HepG2 cell line. Importantly, the lack of effect on non-tumor cells (IEC18 intestinal epithelium cells demonstrates the selectivity of these molecules for cancer cells. Here, we show that the numbers and positions of hydroxy and methoxy groups are crucial for the inhibition efficacy. In addition, the presence of at least one phenolic group is essential for the antitumoral activity. Moreover, in the series of ferrocenylstilbene analogs, the presence of a hidden phenolic function allows for a better solubilization in the cellular environment and significantly increases the antitumoral activity.

  12. Glial cell line-derived neurotrophic factor induced the differentiation of amniotic fluid-derived stem cells into vascular endothelial-like cells in vitro.

    Science.gov (United States)

    Zhang, Ruyu; Lu, Ying; Li, Ju; Wang, Jia; Liu, Caixia; Gao, Fang; Sun, Dong

    2016-02-01

    Amniotic fluid-derived stem cells (AFSCs) are a novel source of stem cells that are isolated and cultured from second trimester amniocentesis. Glial cell line-derived neurotrophic factor (GDNF) acts as a tissue morphogen and regulates stem cell proliferation and differentiation. This study investigated the effect of an adenovirus-mediated GDNF gene, which was engineered into AFSCs, on the cells' biological properties and whether GDNF in combination with AFSCs can be directionally differentiated into vascular endothelial-like cells in vitro. AFSCs were isolated and cultured using the plastic adherence method in vitro and identified by the transcription factor Oct-4, which is the primary marker of pluripotent stem cells. AFSCs were efficiently transfected by a GFP-labeled plasmid system of an adenovirus vector carrying the GDNF gene (Ad-GDNF-GFP). Transfected AFSCs stably expressed GDNF. Transfected AFSCs were cultured in endothelial growth medium-2 containing vascular endothelial growth factor. After 1 week, AFSCs were positive for von Willebrand factor (vWF) and CD31, which are markers of endothelial cells, and the recombinant GDNF group was significantly higher than undifferentiated controls and the GFP only group. These results demonstrated that AFSCs differentiated into vascular endothelial-like cells in vitro, and recombinant GDNF promoted differentiation. The differentiation-induced AFSCs may be used as seed cells to provide a new manner of cell and gene therapies for transplantation into the vascular injury site to promote angiogenesis.

  13. Intraspinal transplantation of motoneuron-like cell combined with delivery of polymer-based glial cell line-derived neurotrophic factor for repair of spinal cord contusion injury

    Institute of Scientific and Technical Information of China (English)

    Alireza Abdanipour; Taki Tiraihi; Taher Taheri

    2014-01-01

    To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord con-tusion injury, we developed rat models of spinal cord contusion injury, 7 days later, injected adipose-derived stem cells-transdifferentiated motoneurons into the epicenter, rostral and caudal regions of the impact site and simultaneously transplanted glial cell line-derived neuro-trophic factor-gelfoam complex into the myelin sheath. Motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery reduced cavity formations and increased cell density in the transplantation site. The combined therapy exhibited superior promoting effects on recovery of motor function to transplantation of glial cell line-derived neurotrophic factor, adipose-derived stem cells or motoneurons alone. These ifndings suggest that motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery holds a great promise for repair of spinal cord injury.

  14. Accelerated senescence and enhanced disease resistance in hybrid chlorosis lines derived from interspecific crosses between tetraploid wheat and Aegilops tauschii.

    Science.gov (United States)

    Nakano, Hiroki; Mizuno, Nobuyuki; Tosa, Yukio; Yoshida, Kentaro; Park, Pyoyun; Takumi, Shigeo

    2015-01-01

    Hybrid chlorosis, a type of hybrid incompatibility, has frequently been reported in inter- and intraspecific crosses of allopolyploid wheat. In a previous study, we reported some types of growth abnormalities such as hybrid necrosis and observed hybrid chlorosis with mild or severe abnormalities in wheat triploids obtained in crosses between tetraploid wheat cultivar Langdon and four Ae. tauschii accessions and in their derived synthetic hexaploids. However, the molecular mechanisms underlying hybrid chlorosis are not well understood. Here, we compared cytology and gene expression in leaves to characterize the abnormal growth in wheat synthetics showing mild and severe chlorosis. In addition, we compared disease resistance to wheat blast fungus. In total 55 and 105 genes related to carbohydrate metabolism and 53 and 89 genes for defense responses were markedly up-regulated in the mild and severe chlorosis lines, respectively. Abnormal chloroplasts formed in the mesophyll cells before the leaves yellowed in the hybrid chlorosis lines. The plants with mild chlorosis showed increased resistance to wheat blast and powdery mildew fungi, although significant differences only in two, third internode length and maturation time, out of the examined agricultural traits were found between the wild type and plants showing mild chlorosis. These observations suggest that senescence might be accelerated in hybrid chlorosis lines of wheat synthetics. Moreover, in wheat synthetics showing mild chlorosis, the negative effects on biomass can be minimized, and they may show substantial fitness under pathogen-polluted conditions.

  15. Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines.

    Science.gov (United States)

    Souza, Raquel P; Bonfim-Mendonça, Patrícia de S; Gimenes, Fabrícia; Ratti, Bianca A; Kaplum, Vanessa; Bruschi, Marcos L; Nakamura, Celso V; Silva, Sueli O; Maria-Engler, Silvya S; Consolaro, Marcia E L

    2017-01-01

    Recently, the cytotoxic effects of apigenin (4',5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

  16. Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

    Directory of Open Access Journals (Sweden)

    Raquel P. Souza

    2017-01-01

    Full Text Available Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone, particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive, SiHa (HPV 16-positive, CaSki (HPV 16 and HPV 18-positive, and C33A (HPV-negative cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT. Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells. Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes.

  17. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    Science.gov (United States)

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, Jürgen J.; Johne, Reimar

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown. PMID:27690085

  18. Anti-proliferation effects of benzimidazole derivatives on HCT-116 colon cancer and MCF-7 breast cancer cell lines.

    Science.gov (United States)

    Al-Douh, Mohammed Hadi; Sahib, Hayder B; Osman, Hasnah; Abd Hamid, Shafida; Salhimi, Salizawati M

    2012-01-01

    Benzimidazoles 1-4 were obtained using modified synthesis methods and studied for their ability to inhibit cell proliferation of colon cancer cell HCT-116 and breast cancer cell MCF-7 using MTT assays. In the HCT-116 cell line, benzimidazole 2 was found to have an IC50 value of 16.2 ± 3.85 μg/mL and benzimidazole 1 a value of 28.5 ± 2.91 μg/mL, while that for benzimidazole 4 was 24.08 ± 0.31 μg/mL. In the MCF-7 cell line, benzimidazole 4 had an IC50 value of 8.86 ± 1.10 μg/mL, benzimidazole 2 a value of 30.29 ± 6.39 μg/mL, and benzimidazole 1 a value of 31.2 ± 4.49 μg/mL. Benzimidazole 3 exerted no cytotoxicity in either of the cell lines, with IC50 values >50 μg/mL. The results suggest that benzimidazoles derivatives may have chemotherapeutic potential for treatment of both colon and breast cancers.

  19. Accelerated senescence and enhanced disease resistance in hybrid chlorosis lines derived from interspecific crosses between tetraploid wheat and Aegilops tauschii.

    Directory of Open Access Journals (Sweden)

    Hiroki Nakano

    Full Text Available Hybrid chlorosis, a type of hybrid incompatibility, has frequently been reported in inter- and intraspecific crosses of allopolyploid wheat. In a previous study, we reported some types of growth abnormalities such as hybrid necrosis and observed hybrid chlorosis with mild or severe abnormalities in wheat triploids obtained in crosses between tetraploid wheat cultivar Langdon and four Ae. tauschii accessions and in their derived synthetic hexaploids. However, the molecular mechanisms underlying hybrid chlorosis are not well understood. Here, we compared cytology and gene expression in leaves to characterize the abnormal growth in wheat synthetics showing mild and severe chlorosis. In addition, we compared disease resistance to wheat blast fungus. In total 55 and 105 genes related to carbohydrate metabolism and 53 and 89 genes for defense responses were markedly up-regulated in the mild and severe chlorosis lines, respectively. Abnormal chloroplasts formed in the mesophyll cells before the leaves yellowed in the hybrid chlorosis lines. The plants with mild chlorosis showed increased resistance to wheat blast and powdery mildew fungi, although significant differences only in two, third internode length and maturation time, out of the examined agricultural traits were found between the wild type and plants showing mild chlorosis. These observations suggest that senescence might be accelerated in hybrid chlorosis lines of wheat synthetics. Moreover, in wheat synthetics showing mild chlorosis, the negative effects on biomass can be minimized, and they may show substantial fitness under pathogen-polluted conditions.

  20. A new permanent cell line derived from the bank vole (Myodes glareolus as cell culture model for zoonotic viruses

    Directory of Open Access Journals (Sweden)

    Herzog Sibylle

    2011-07-01

    Full Text Available Abstract Background Approximately 60% of emerging viruses are of zoonotic origin, with three-fourths derived from wild animals. Many of these zoonotic diseases are transmitted by rodents with important information about their reservoir dynamics and pathogenesis missing. One main reason for the gap in our knowledge is the lack of adequate cell culture systems as models for the investigation of rodent-borne (robo viruses in vitro. Therefore we established and characterized a new cell line, BVK168, using the kidney of a bank vole, Myodes glareolus, the most abundant member of the Arvicolinae trapped in Germany. Results BVK168 proved to be of epithelial morphology expressing tight junctions as well as adherence junction proteins. The BVK168 cells were analyzed for their infectability by several arbo- and robo-viruses: Vesicular stomatitis virus, vaccinia virus, cowpox virus, Sindbis virus, Pixuna virus, Usutu virus, Inkoo virus, Puumalavirus, and Borna disease virus (BDV. The cell line was susceptible for all tested viruses, and most interestingly also for the difficult to propagate BDV. Conclusion In conclusion, the newly established cell line from wildlife rodents seems to be an excellent tool for the isolation and characterization of new rodent-associated viruses and may be used as in vitro-model to study properties and pathogenesis of these agents.

  1. A Comparitive Assessement of Cytokine Expression in Human-Derived Cell Lines Exposed to Alpha Particles and X-Rays

    Directory of Open Access Journals (Sweden)

    Vinita Chauhan

    2012-01-01

    Full Text Available Alpha- (α- particle radiation exposure has been linked to the development of lung cancer and has been identified as a radiation type likely to be employed in radiological dispersal devices. Currently, there exists a knowledge gap concerning cytokine modulations associated with exposure to α-particles. Bio-plex technology was employed to investigate changes in proinflammatory cytokines in two human-derived cell lines. Cells were irradiated at a dose of 1.5 Gy to either α-particles or X-rays at equivalent dose rates. The two cell lines exhibited a unique pattern of cytokine expression and the response varied with radiation type. Of the 27 cytokines assessed, only vascular endothelin growth factor (VEGF was observed to be modulated in both cell lines solely after α-particle exposure, and the expression of VEGF was shown to be dose responsive. These results suggest that certain proinflammatory cytokines may be involved in the biological effects related to α- particle exposure and the responses are cell type and radiation type specific.

  2. Influence of Spray-dried Hydroxyapatite-5-Fluorouracil Granules on Cell Lines Derived from Tissues of Mesenchymal Origin

    Directory of Open Access Journals (Sweden)

    Tim Scharnweber

    2008-11-01

    Full Text Available In our previous work we described the preparation and characterization of spray dried hydroxyapatite micro granules loaded with 5-fluorouracil (5-FU. These loaded particles are used as a model drug delivery system (DDS. In this study we examined the in vitro response of two cell lines derived from different tissues to 5-FU loaded granules (LG. Both cell lines, either L929 cells of a mouse fibroblast lineage or cells originating from a rat osteosarcoma (ROS 17/2.8 showed a dose dependent decrease in cell proliferation in response to 5-FU-, either dissolved in the culture medium or loaded onto particles. The response of the two cell lines to loaded and nonloaded particles was different. The effect of LG and of a corresponding concentration of free 5-FU was practically the same for the ROS 17/2.8 cells indicating that ROS 17/2.8 cells were not affected by the carrier material. In contrast, L929 cells showed a slight decrease in cell proliferation also in the presence of granules not loaded with 5-FU. This is thought to be attributed to the inhibition of mitogenesis by phosphocitrates, already demonstrated in fibroblasts. In summary, we found that the loaded 5-FU kept its effectivity after the spray drying process and that the response towards the granules varied with cell type. This is the first step towards a tissue specific DDS.

  3. Oxidative Stress Triggered by Apigenin Induces Apoptosis in a Comprehensive Panel of Human Cervical Cancer-Derived Cell Lines

    Science.gov (United States)

    Souza, Raquel P.; Gimenes, Fabrícia; Ratti, Bianca A.; Kaplum, Vanessa; Bruschi, Marcos L.; Nakamura, Celso V.; Maria-Engler, Silvya S.

    2017-01-01

    Recently, the cytotoxic effects of apigenin (4′,5,7-trihydroxyflavone), particularly its marked inhibition of cancer cell viability both in vitro and in vivo, have attracted the attention of the anticancer drug discovery field. Despite this, there are few studies of apigenin in cervical cancer, and these studies have mostly been conducted using HeLa cells. To evaluate the possibility of apigenin as a new therapeutic candidate for cervical cancer, we evaluated its cytotoxic effects in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16 and HPV 18-positive), and C33A (HPV-negative) cells in comparison to a nontumorigenic spontaneously immortalized human epithelial cell line (HaCaT). Our results demonstrated that apigenin had a selective cytotoxic effect and could induce apoptosis in all cervical cancer cell lines which were positively marked with Annexin V, but not in HaCaT (control cells). Additionally, apigenin was able to induce mitochondrial redox impairment, once it increased ROS levels and H2O2, decreased the Δψm, and increased LPO. Still, apigenin was able to inhibit migration and invasion of cancer cells. Thus, apigenin appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV genotypes. PMID:28191273

  4. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    Science.gov (United States)

    Solarte, Víctor A.; Rosas, Jaiver E.; Rivera, Zuly J.; Arango-Rodríguez, Martha L.; García, Javier E.; Vernot, Jean-Paul

    2015-01-01

    Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC. PMID:26609531

  5. A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Víctor A. Solarte

    2015-01-01

    Full Text Available Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–254, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90% in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

  6. Induction of cell death by ascorbic acid derivatives in human renal carcinoma and glioblastoma cell lines.

    Science.gov (United States)

    Makino, Y; Sakagami, H; Takeda, M

    1999-01-01

    Sodium-L-ascorbate, L-ascorbic acid, D-isoascorbic acid, sodium 5,6-benzylidene-L-ascorbate and sodium-6-beta-O-galactosyl-L-ascorbate, which produce ascorbyl radicals during the oxidative degradation, also induced cytotoxicity against cultured human renal carcinoma (TC-1) and glioblastoma multiform tumor (T98G) cell lines. On the other hand, L-ascorbic acid 2-phosphate magnesium and L-ascorbic acid 2-sulfate dipotassium salt, which do not produce the ascorbyl radical, were inactive. This suggests the possible role of the ascorbyl radical for cell death induction. T98G cells were more resistant to ascorbate analogs than TC-1 and HL-60 cells, possibly due to higher intracellular glutathione concentrations. Ascorbate treatment induced rapid elevation of both intracellular concentration of cAMP and Ca2+ in HL-60 cells, but not in TC-1 and T98G cells. However, the elevation of cAMP by theophyline and N,2-dibutyryl adenosine 3,5 cyclic monophosphate (dibutyryl cAMP) resulted in a decrease in the viable cell number. This suggests the possible role of cAMP for ascorbate-induced cell death.

  7. Mapping of extreme resistance to PVY (Ry (sto)) on chromosome XII using anther-culture-derived primary dihaploid potato lines.

    Science.gov (United States)

    Song, Ye-Su; Hepting, Leonard; Schweizer, Günther; Hartl, Lorenz; Wenzel, Gerhard; Schwarzfischer, Andrea

    2005-09-01

    The inheritance of extreme resistance to PVY (Ry (sto)) by a single dominant locus was confirmed by obtaining a 1:1 segregation ratio in a virus inoculation test with 28 resistant (Ryry) to 29 susceptible (ryry) anther culture-derived dihaploid lines (2n=2x=24) from cv. "Assia" (2n=4x=48) having extreme resistance derived from Solanum stoloniferum in simplex constitution (Ryryryry). Twelve Ry (sto) markers selected in AFLP assays using bulked segregant analysis were applied to 106 tested potato cultivars from Germany, The Netherlands and Poland and 19 potato cultivars were identified by these markers as extremely resistant to PVY in alignment with phenotypic data. The locus for extreme resistance (Ry (sto)) to PVY was mapped on chromosome XII co-segregating with the SSR marker STM 0003. The utility of anther-culture derived dihaploid potatoes for genetic marker development was demonstrated. Marker transferability from diploids to tetraploids provides an optimistic potential for marker-assisted selection in potato breeding programs.

  8. Susceptibility Analysis of a Cell Line Derived from Aedes aegypti (Diptera: ulicidae

    Directory of Open Access Journals (Sweden)

    Carolina Muñoz-Camargo

    2005-12-01

    Full Text Available The susceptibility of culture cells derived fromembryonic tissues of Aedes aegypti to theinfection with Leishmania (L chagasi andLeishmania (V braziliensis was evaluated.Methodology: These parasites are etiologicalagents of American visceral leishmaniasis andcutaneous leishmaniasis, respectively. Selectedcells of Aedes aegypti were maintained in culturemedium Grace/L15, supplement with 15% bovinefetal serum, 5,4 mg/ml of albendazol and anantibiotic mixture and incubated at an average temperature of 26°C. The cultures were inoculatedwith metacyclic promastigotes of the strain MH/CO/84/CI-044B of L. chagasi and the strainHOM/BR752903 of L. braziliensis in a concentrationof 10 parasites by cell. The J774 cellline was used as positive control of infection.Results: The highest percentage of infectionrepresented as the number of amastigotes per cellin A. aegyti cell cultures and in the J774 cell linewere obtained on days 6 and 9 post-infection.The results showed interaction, internalizationand maturation in vitro of the two species of theparasite in the cells of a non-vector insect ofLeishmania. Infected A. aegypti cells showedchanges in its area because of the influence of theparasites that differ significantly (P <0.05compared to not infected cells. Conclusion: Cellcultures from A. aegypti emerge as a new in vitromodel for the study of the biological cycle of L.chagasi and L. braziliensis.

  9. Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

  10. Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

    Science.gov (United States)

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state. PMID:25680105

  11. Effects of Simulated Microgravity on the Expression Profile of Microrna in Human Lymphoblastoid Cells

    Science.gov (United States)

    Zhang, Ye; Wu, Honglu; Ramesh, Govindarajan; Rohde, Larry; Story, Michael; Mangala, Lingegowda

    2012-07-01

    EFFECTS OF SIMULATED MICROGRAVITY ON THE EXPRESSION PROFILE OF MICRORNA IN HUMAN LYMPHOBLASTOID CELLS Lingegowda S. Mangala1,2, Ye Zhang1,3, Zhenhua He2, Kamal Emami1, Govindarajan T. Ramesh4, Michael Story 5, Larry H. Rohde2, and Honglu Wu1 1 NASA Johnson Space Center, Houston, Texas, USA 2 University of Houston Clear Lake, Houston, Texas, USA 3 Wyle Integrated Science and Engineering Group, Houston, Texas, USA 4 Norfolk State University, Norfolk, VA, USA 5 University of Texas, Southwestern Medical Center, Dallas, Texas, USA This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison to static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a High Aspect Ratio Vessel (HARV; bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNA was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22 and miR-141, miR-618 and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using q-RT PCR. Network and pathway analysis of gene and miRNA expression profiles indicates that the regulation of cell communication and catalytic activities, as well as pathways involved in immune response_IL-15

  12. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Science.gov (United States)

    Hart, Felix; Danielczyk, Antje; Goletz, Steffen

    2017-01-01

    IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich) and hematological (CD20) cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  13. Muscarinic receptors stimulate cell proliferation in the human urothelium-derived cell line UROtsa.

    Science.gov (United States)

    Arrighi, Nicola; Bodei, Serena; Lucente, Alessandra; Michel, Martin C; Zani, Danilo; Simeone, Claudio; Cunico, Sergio Cosciani; Spano, PierFranco; Sigala, Sandra

    2011-10-01

    The widespread non-neuronal synthesis of acetylcholine (ACh) has changed the paradigm of ACh acting solely as a neurotransmitter. Indeed, the presence of ACh in many types of proliferating cells suggests a role for this neurotransmitter in the control of cell division. The parasympathetic system is a major pathway regulating micturition, but ACh-mediated control plays a more complex role than previously described, acting not only in the detrusor muscle, but also influencing detrusor function through the activity of urothelial muscarinic receptors. Here we investigated the role of muscarinic receptors in mediating cell proliferation in the human UROtsa cell line, which is a widely used experimental model to study urothelium physiology and pathophysiology. Our results demonstrate that UROtsa cells express the machinery for ACh synthesis and that muscarinic receptors, with the rank order of M3>M2>M5>M1=M4, are present and functionally linked to their known second messengers. Indeed, the cholinergic receptor agonist carbachol (CCh) (1-100 μM) concentration-dependently raised IP(3) levels, reaching 66±5% over basal. The forskolin-mediated adenylyl cyclase activation was reduced by CCh exposure (forskolin: 1.4±0.14 pmol/ml; forskolin+100 μM CCh: 0.84±0.12 pmol/ml). CCh (1-100 μM) concentration-dependently increased UROtsa cell proliferation and this effect was inhibited by the non-selective antagonist atropine and the M(3)-selective antagonists darifenacin and J104129. Finally, CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation.

  14. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

    Directory of Open Access Journals (Sweden)

    Felix Hart

    2017-05-01

    Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  15. Genome-wide association of 10 horticultural traits with expressed sequence tag-derived SNP markers in a collection of lettuce lines

    Science.gov (United States)

    Genetic diversity, population structure, and genome-wide marker-trait association analyses were conducted on a special collection of 298 homozygous lettuce (Lactuca sativa L.) lines. Each of these lines was derived from a single plant that had been genotyped with 384 SNP makers using LSGermOPA. They...

  16. Canine distemper virus induces apoptosis in cervical tumor derived cell lines

    Directory of Open Access Journals (Sweden)

    Rajão Daniela S

    2011-06-01

    Full Text Available Abstract Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi, by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

  17. Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

    Science.gov (United States)

    Beckman, I G; Bradley, J; Brooks, D A; Kupa, A; McNamara, P J; Thomas, M E; Zola, H

    1980-06-01

    A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.

  18. Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

    Directory of Open Access Journals (Sweden)

    López Jacqueline

    2012-01-01

    Full Text Available Abstract Background Cancer-initiating cells (CICs are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I. Methods Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP. Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions. Results CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 103 dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 105 monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB DNA repair machinery and the metabolism of reactive oxygen species (ROS. Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR. Conclusions We characterized a self-renewing subpopulation of CICs found among

  19. Secretion of nerve growth factor, brain-derived neurotrophic factor, and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium

    Institute of Scientific and Technical Information of China (English)

    Sanjiang Feng; Minghua Zhuang; Rui Wu

    2012-01-01

    The present study co-cultured human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium- containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7–10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid (10%, 20% and 30%) in the growth medium caused a decrease of neurotrophic factor secretion. Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.

  20. Omega 3 fatty acids increase the chemo-sensitivity of B-CLL-derived cell lines EHEB and MEC-2 and of B-PLL-derived cell line JVM-2 to anti-cancer drugs doxorubicin, vincristine and fludarabine.

    Science.gov (United States)

    Fahrmann, Johannes F; Hardman, W Elaine

    2013-03-16

    B-Cell chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the United States. Clinical treatment of CLL is often limited due to drug resistance and severe therapy-induced toxicities. We hypothesized that the omega 3 (n-3) fatty acids, eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA), would increase the sensitivity of malignant B-lymphocytes to anti-cancer drugs doxorubicin, vincristine and/or fludarabine in vitro and that increased sensitivity is achieved by alterations in cell-cycle progression leading to growth inhibition and/or enhanced cell death. We further postulate that enhanced sensitivity is dependent on the formation of lipid peroxides and to the generation of reactive oxygen species (ROS). In the present study, B-CLL-derived leukemic cell lines EHEB and MEC-2 and the B-Prolymphocytic leukemic-derived (PLL) cell line JVM-2 were tested for in vitro sensitivity against doxorubicin, vincristine or fludarabine in the presence or absence of vehicle, arachidonic acid (omega 6), EPA or DHA. Cell cycle analysis and Annexin-V assays were performed to determine cell cycle progression and % apoptotic cells, respectively. Assays for malondialdehyde, a measure of lipid peroxidation, and DCF fluorescence assays, a measure of intracellular ROS, were performed to determine if enhanced sensitivity of cells to the drugs by n-3 was dependent on the formation of ROS. Our results indicated that: 1) EPA and DHA differentially sensitized B-leukemic cell lines EHEB, JVM-2 and MEC-2 to doxorubicin, vincristine and fludarabine in vitro; 2) n-3 alone and with drug treatment increased cell death and induced G2/M arrest in a cell-type specific manner; 3) lipid peroxidation increased in the presence of n-3; 4) there was higher lipid peroxidation in MEC-2 cells in presence of DHA and doxorubicin than with either alone; 5) n-3 increased generation of ROS in MEC-2, and 6) the addition of vitamin-E abrogated the increase in ROS generation and chemo

  1. Alterations in BDNF (brain derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) serum levels in bipolar disorder: The role of lithium.

    Science.gov (United States)

    Tunca, Zeliha; Ozerdem, Aysegul; Ceylan, Deniz; Yalçın, Yaprak; Can, Güneş; Resmi, Halil; Akan, Pınar; Ergör, Gül; Aydemir, Omer; Cengisiz, Cengiz; Kerim, Doyuran

    2014-09-01

    Brain-derived neurotrophic factor (BDNF) has been consistently reported to be decreased in mania or depression in bipolar disorders. Evidence suggests that Glial cell line-derived neurotrophic factor (GDNF) has a role in the pathogenesis of mood disorders. Whether GDNF and BDNF act in the same way across different episodes in bipolar disorders is unclear. BDNF and GDNF serum levels were measured simultaneously by enzyme-linked immunosorbent assay (ELISA) method in 96 patients diagnosed with bipolar disorder according to DSM-IV (37 euthymic, 33 manic, 26 depressed) in comparison to 61 healthy volunteers. SCID- I and SCID-non patient version were used for clinical evaluation of the patients and healthy volunteers respectively. Correlations between the two trophic factor levels, and medication dose, duration and serum levels of lithium or valproate were studied across different episodes of illness. Patients had significantly lower BDNF levels during mania and depression compared to euthymic patients and healthy controls. GDNF levels were not distinctive. However GDNF/BDNF ratio was higher in manic state compared to euthymia and healthy controls. Significant negative correlation was observed between BDNF and GDNF levels in euthymic patients. While BDNF levels correlated positively, GDNF levels correlated negatively with lithium levels. Regression analysis confirmed that lithium levels predicted only GDNF levels positively in mania, and negatively in euthymia. Small sample size in different episodes and drug-free patients was the limitation of thestudy. Current data suggests that lithium exerts its therapeutic action by an inverse effect on BDNF and GDNF levels, possibly by up-regulating BDNF and down-regulating GDNF to achieve euthymia. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Low Dose Radiation Response Curves, Networks and Pathways in Human Lymphoblastoid Cells Exposed from 1 to 10 cGy of Acute Gamma Radiation

    Energy Technology Data Exchange (ETDEWEB)

    Wyrobek, A. J.; Manohar, C. F.; Nelson, D. O.; Furtado, M. R.; Bhattacharya, M. S.; Marchetti, F.; Coleman, M.A.

    2011-04-18

    We investigated the low dose dependency of the transcriptional response of human cells to characterize the shape and biological functions associated with the dose response curve and to identify common and conserved functions of low dose expressed genes across cells and tissues. Human lymphoblastoid (HL) cells from two unrelated individuals were exposed to graded doses of radiation spanning the range of 1-10 cGy were analyzed by transcriptome profiling, qPCR and bioinformatics, in comparison to sham irradiated samples. A set of {approx}80 genes showed consistent responses in both cell lines; these genes were associated with homeostasis mechanisms (e.g., membrane signaling, molecule transport), subcellular locations (e.g., Golgi, and endoplasmic reticulum), and involved diverse signal transduction pathways. The majority of radiation-modulated genes had plateau-like responses across 1-10 cGy, some with suggestive evidence that transcription was modulated at doses below 1 cGy. MYC, FOS and TP53 were the major network nodes of the low-dose response in HL cells. Comparison our low dose expression findings in HL cells with those of prior studies in mouse brain after whole body exposure, in human keratinocyte cultures, and in endothelial cells cultures, indicates that certain components of the low dose radiation response are broadly conserved across cell types and tissues, independent of proliferation status.

  3. P53 alters the cytotoxicity and genotoxicity for oxidized graphene in human B-lymphoblastoid cells

    Science.gov (United States)

    Petibone, Dayton Matthew

    Widespread use of oxidized graphene nanomaterials in industry, medicine, and consumer products raises concern about potential adverse impacts on human health. The p53 tumor suppressor protein is crucial to maintaining cellular and genetic stability to prevent carcinogenesis. Here, we show that oxygen functionalized graphene (f-G) absorption and p53 functional status correlate with cytotoxicity and genotoxicity in human B-lymphoblastoid cells. Trends in f-G absorption by were dose-dependent. Cells with functional p53 exposed to f-G arrested in G0/G1 phase of the cell cycle, suppressed f-G induced reactive oxygen species (ROS), and had elevated apoptosis. While compared to p53 competent cells, the p53 deficient cells exposed to f-G accumulated in S-phase of the cell cycle, had elevated ROS levels, and evaded apoptosis. The f-G genotoxicity was evident as increased loss-of-heterozygosity mutants independent of p53 status, and structural chromosome damage in p53 deficient cells. These findings have broad implications for the safety and efficacy of oxidized graphene nanomaterials in industrial, consumer products and biomedical applications.

  4. Cold Atmospheric Plasma Induces Apoptosis and Oxidative Stress Pathway Regulation in T-Lymphoblastoid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Eleonora Turrini

    2017-01-01

    Full Text Available Cold atmospheric plasma (CAP has shown its antitumor activity in both in vitro and in vivo systems. However, the mechanisms at the basis of CAP-cell interaction are not yet completely understood. The aim of this study is to investigate CAP proapoptotic effect and identify some of the molecular mechanisms triggered by CAP in human T-lymphoblastoid leukemia cells. CAP treatment was performed by means of a wand electrode DBD source driven by nanosecond high-voltage pulses under different operating conditions. The biological endpoints were assessed through flow cytometry and real-time PCR. CAP caused apoptosis in Jurkat cells mediated by p53 upregulation. To test the involvement of intrinsic and/or extrinsic pathway, the expression of Bax/Bcl-2 and caspase-8 was analyzed. The activation of caspase-8 and the upregulation of Bax and Bcl-2 were observed. Moreover, CAP treatment increased ROS intracellular level. The situation reverts after a longer time of treatment. This is probably due to compensatory cellular mechanisms such as the posttranscriptional upregulation of SOD1, CAT, and GSR2. According to ROS increase, CAP induced a significant increase in DNA damage at all treatment conditions. In conclusion, our results provide a deeper understanding of CAP potential in the oncological field and pose the basis for the evaluation of its toxicological profile.

  5. Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells.

    Science.gov (United States)

    Masci, Alessandra; Mastronicola, Daniela; Arese, Marzia; Piane, Maria; De Amicis, Andrea; Blanck, Thomas J J; Chessa, Luciana; Sarti, Paolo

    2008-01-01

    Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P< or =0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.

  6. siRNA targeting stathmin inhibits invasion and enhances chemotherapy sensitivity of stem cells derived from glioma cell lines.

    Science.gov (United States)

    Song, Yuwen; Mu, Luyan; Han, Xuezhe; Liu, Xiaoqian; Fu, Songbin

    2014-12-01

    Glioma is one of the most highly angiogenic tumors, and glioma stem cells (GSCs) are responsible for resistance to chemotherapy and radiotherapy, as well as recurrence after operation. Stathmin is substantial for mitosis and plays an important role in proliferation and migration of glioma-derived endothelial cells. However, the relationship between stathmin and GSCs is incompletely understood. Here we isolated GSCs from glioma cell lines U87MG and U251, and then used siRNA targeting stathmin for silencing. We showed that silencing of stathmin suppressed the proliferation, increased the apoptosis rate, and arrested the cell cycle at G2/M phase in GSCs. Silencing of stathmin in GSCs also resulted in inhibited the migration/invasion as well as the capability of vasculogenic mimicry. The susceptibility of GSCs to temozolomide was also enhanced by stathmin silencing. Our findings suggest stathmin as a potential target in GSCs for glioma treatment.

  7. Sequence analysis and functional study of the Han Nationality glial cell line-derived neurotrophic factor transcript

    Institute of Scientific and Technical Information of China (English)

    CHEN Zhe-yu; HUANG Ai-jun; LU Chang-lin; WU Xiang-fu; HE Cheng

    2001-01-01

    To study the sequence and function of the glial cell line-derived neurotrophic factor (GDNF) transcript in subjects of Han nationality. Methods: The Han nationality GDNF transcript was amplified by RT-PCR and expressed by baculovirus expression system. Biological activity of the expressed product was measured by the primary culture of midbrain dopaminergic neurons. Results: There only existed the shorter GDNF transcript of 555 bp in the Han nationality. The secretory expression product of the shorter transcript in insect cells promoted the survival and differentiation of dopaminergic neurons. Conclusion: It is found that there is a 78 bp deletion in the Han nationality GDNF transcript compared with the reported 633 bp GDNF transcript. The 78 bp deletion does not affect the secretory expression and biological activity of GDNF mature protein.

  8. Glial cell line-derived neurotrophic factor (GDNF) expression and NMJ plasticity in skeletal muscle following endurance exercise.

    Science.gov (United States)

    Gyorkos, A M; McCullough, M J; Spitsbergen, J M

    2014-01-17

    Glial cell line-derived neurotrophic factor (GDNF) supports and maintains the neuromuscular system during development and through adulthood by promoting neuroplasticity. The aim of this study was to determine if different modes of exercise can promote changes in GDNF expression and neuromuscular junction (NMJ) morphology in slow- and fast-twitch muscles. Rats were randomly assigned to a run training (run group), swim training (swim group), or sedentary control group. GDNF protein content was determined by enzyme-linked immunosorbant assay. GDNF protein content increased significantly in soleus (SOL) following both training protocols (PGDNF content and total end plate area were positively correlated. End plate area decreased in EDL of the run group and increased in SOL of the swim group. The results indicate that GDNF expression and NMJ morphological changes are activity dependent and that different changes may be observed by varying the exercise intensity in slow- and fast-twitch fibers.

  9. Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes.

    Directory of Open Access Journals (Sweden)

    Lara Stevanato

    Full Text Available Exosomes are small (30-100 nm membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs. Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP, and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369. By using next generation sequencing (NGS technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246 in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3' untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.

  10. New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line.

    Science.gov (United States)

    Terawaki, Kiyoshi; Sawada, Yumi; Kashiwase, Yohei; Hashimoto, Hirofumi; Yoshimura, Mitsuhiro; Suzuki, Masami; Miyano, Kanako; Sudo, Yuka; Shiraishi, Seiji; Higami, Yoshikazu; Yanagihara, Kazuyoshi; Kase, Yoshio; Ueta, Yoichi; Uezono, Yasuhito

    2014-02-15

    Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

  11. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.

    Science.gov (United States)

    Andrews, P W; Damjanov, I; Simon, D; Banting, G S; Carlin, C; Dracopoli, N C; Føgh, J

    1984-02-01

    We have derived and characterized single cell clones from a xenograft tumor of the teratocarcinoma cell line Tera-2. Isozyme and chromosomal analyses confirmed their common origin. When cultures of the clones were maintained at a high cell density, many cells exhibited a morphology and cell surface antigen phenotype typical of human embryonal carcinoma cells. These features included a high nucleo-cytoplasmic ratio, prominent nucleoli, and the expression of the globoseries glycolipid antigen SSEA-3. In addition, other cells, in many respects resembling these typical embryonal carcinoma cells, were distinguished by a marked tendency to accumulate cytoplasmic glycogen. Similar cells, together with more differentiated cells, were seen in low passage cultures of Tera-2 itself. When the clones were grown at a low cell density many cells assumed a larger, flatter shape, a few with multiple nucleoli. Also, the fucosylated lactosamine antigen SSEA-1 appeared on some cells, whereas expression of SSEA-3 and HLA-A,B,C tended to be reduced. Often the synthesis of fibronectin was increased. However, no obvious cytoplasmic differentiation was seen upon ultrastructural examination, and synthesis of human chorionic gonadotropin, alpha-fetoprotein, and laminin was not detected. In contrast to the limited spontaneous changes seen in culture, marked differentiation occurred in tumors obtained following injection of the cells into athymic (nu/nu) mice. In additional to embryonal carcinoma cells, these tumors contained a variety of somatic tissues that included glandular structures, possibly related to the primitive gut, and neural elements. These cell lines derived from Tera-2 constitute the first example of clonal human embryonal carcinoma cells, adapted to growth in vitro, that have retained the capacity for differentiation into diverse somatic tissues.

  12. Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes.

    Science.gov (United States)

    Stevanato, Lara; Thanabalasundaram, Lavaniya; Vysokov, Nickolai; Sinden, John D

    2016-01-01

    Exosomes are small (30-100 nm) membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs) are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs). Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP), and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369). By using next generation sequencing (NGS) technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246) in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3' untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.

  13. Association between serum levels of glial cell-line derived neurotrophic factor and attention deficits in schizophrenia.

    Science.gov (United States)

    Niitsu, Tomihisa; Shirayama, Yukihiko; Matsuzawa, Daisuke; Shimizu, Eiji; Hashimoto, Kenji; Iyo, Masaomi

    2014-07-11

    Several lines of evidence suggest that glial cell-line derived neurotrophic factor (GDNF) plays an important role in the pathophysiology of neuropsychiatric and neurodegenerative disorders. In this study, we investigated the association between GDNF serum levels and the clinical status of medicated patients with schizophrenia. Sixty-three medicated patients with schizophrenia and 52 age- and sex-matched healthy controls were recruited. Patients were evaluated using the brief psychiatry rating scale, the scale for the assessment of negative symptoms (SANS) and neuropsychological tests. Serum levels of GDNF were determined using an ELISA method. Serum levels of GDNF did not differ between schizophrenia patients and controls. Higher GDNF serum levels were associated with better performances on the Digit Span in healthy controls but not in schizophrenics. At the same time, higher GDNF serum levels were associated with severe attention deficits on the SANS subscale, in schizophrenics. Our preliminary study suggests that serum levels of GDNF may be an unsuitable biomarker for schizophrenia, although it may be associated with working memory in healthy controls and the pathophysiology of attention deficits in schizophrenia.

  14. QTL mapping of agronomic waterlogging tolerance using recombinant inbred lines derived from tropical maize (Zea mays L) germplasm.

    Science.gov (United States)

    Zaidi, Pervez Haider; Rashid, Zerka; Vinayan, Madhumal Thayil; Almeida, Gustavo Dias; Phagna, Ramesh Kumar; Babu, Raman

    2015-01-01

    Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate

  15. Chordoma-derived cell line U-CH1-N recapitulates the biological properties of notochordal nucleus pulposus cells.

    Science.gov (United States)

    Fujita, Nobuyuki; Suzuki, Satoshi; Watanabe, Kota; Ishii, Ken; Watanabe, Ryuichi; Shimoda, Masayuki; Takubo, Keiyo; Tsuji, Takashi; Toyama, Yoshiaki; Miyamoto, Takeshi; Horiuchi, Keisuke; Nakamura, Masaya; Matsumoto, Morio

    2016-08-01

    Intervertebral disc degeneration proceeds with age and is one of the major causes of lumbar pain and degenerative lumbar spine diseases. However, studies in the field of intervertebral disc biology have been hampered by the lack of reliable cell lines that can be used for in vitro assays. In this study, we show that a chordoma-derived cell line U-CH1-N cells highly express the nucleus pulposus (NP) marker genes, including T (encodes T brachyury transcription factor), KRT19, and CD24. These observations were further confirmed by immunocytochemistry and flow cytometry. Reporter analyses showed that transcriptional activity of T was enhanced in U-CH1-N cells. Chondrogenic capacity of U-CH1-N cells was verified by evaluating the expression of extracellular matrix (ECM) genes and Alcian blue staining. Of note, we found that proliferation and synthesis of chondrogenic ECM proteins were largely dependent on T in U-CH1-N cells. In accordance, knockdown of the T transcripts suppressed the expression of PCNA, a gene essential for DNA replication, and SOX5 and SOX6, the master regulators of chondrogenesis. On the other hand, the CD24-silenced cells showed no reduction in the mRNA expression level of the chondrogenic ECM genes. These results suggest that U-CH1-N shares important biological properties with notochordal NP cells and that T plays crucial roles in maintaining the notochordal NP cell-like phenotype in this cell line. Taken together, our data indicate that U-CH1-N may serve as a useful tool in studying the biology of intervertebral disc. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1341-1350, 2016.

  16. Genotype × Environment Interactions of Yield Traits in Backcross Introgression Lines Derived from Oryza sativa cv. Swarna/Oryza nivara

    Science.gov (United States)

    Balakrishnan, Divya; Subrahmanyam, Desiraju; Badri, Jyothi; Raju, Addanki Krishnam; Rao, Yadavalli Venkateswara; Beerelli, Kavitha; Mesapogu, Sukumar; Surapaneni, Malathi; Ponnuswamy, Revathi; Padmavathi, G.; Babu, V. Ravindra; Neelamraju, Sarla

    2016-01-01

    Advanced backcross introgression lines (BILs) developed from crosses of Oryza sativa var. Swarna/O. nivara accessions were grown and evaluated for yield and related traits. Trials were conducted for consecutive three seasons in field conditions in a randomized complete block design with three replications. Data on yield traits under irrigated conditions were analyzed using the Additive Main Effect and Multiplicative Interaction (AMMI), Genotype and Genotype × Environment Interaction (GGE) and modified rank-sum statistic (YSi) for yield stability. BILs viz., G3 (14S) and G6 (166S) showed yield stability across the seasons along with high mean yield performance. G3 is early in flowering with high yield and has good grain quality and medium height, hence could be recommended for most of the irrigated locations. G6 is a late duration genotype, with strong culm strength, high grain number and panicle weight. G6 has higher yield and stability than Swarna but has Swarna grain type. Among the varieties tested DRRDhan 40 and recurrent parent Swarna showed stability for yield traits across the seasons. The component traits thousand grain weight, panicle weight, panicle length, grain number and plant height explained highest genotypic percentage over environment and interaction factors and can be prioritized to dissect stable QTLs/ genes. These lines were genotyped using microsatellite markers covering the entire rice genome and also using a set of markers linked to previously reported yield QTLs. It was observed that wild derived lines with more than 70% of recurrent parent genome were stable and showed enhanced yield levels compared to genotypes with higher donor genome introgressions.

  17. Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3.

    Science.gov (United States)

    Bratoeff, Eugene; Garrido, Mariana; Ramírez-Apan, Teresa; Heuze, Yvonne; Sánchez, Araceli; Soriano, Juan; Cabeza, Marisa

    2014-11-01

    It is well known that testosterone (T) under the influence of 5α-reductase enzyme is converted to dihydrotestosterone (DHT), which causes androgen-dependent diseases. The aim of this study was to synthesize new dehydroepiandrosterone derivatives (3a-e, 4a-i, 6 and 7) having potential inhibitory activity against the 5α-reductase enzyme. This paper also reports the in vivo pharmacological effect of these steroidal molecules. The results from this study showed that all compounds exhibited low inhibitory activity for 5α-reductase type 1 and 2 enzymes and they failed to bind to the androgen receptor. Furthermore, in the in vivo experiment, steroids 3b, 4f, and 4 g showed comparable antiandrogenic activity to that of finasteride; only derivatives 4d and 7 produced a considerable decrease in the weight of the prostate gland of gonadectomized hamsters treated with (T). On the other hand, compounds 4a, f and h showed 100% inhibition of the growth of prostate cancer cell line PC-3, with compound 4 g having a 98.2% antiproliferative effect at 50 μM. The overall data indicated that these steroidal molecules, having an aromatic ester moiety at C-3 (4f-h), could have anticancer properties.

  18. Continuum representation of systems of dislocation lines: A general method for deriving closed-form evolution equations

    Science.gov (United States)

    Monavari, Mehran; Sandfeld, Stefan; Zaiser, Michael

    2016-10-01

    Plasticity is governed by the evolution of, in general anisotropic, systems of dislocations. We seek to faithfully represent this evolution in terms of density-like variables which average over the discrete dislocation microstructure. Starting from T. Hochrainer's continuum theory of dislocations (CDD) (Hochrainer, 2015), we introduce a methodology based on the 'Maximum Information Entropy Principle' (MIEP) for deriving closed-form evolution equations for dislocation density measures of different order. These equations provide an optimum representation of the kinematic properties of systems of curved and connected dislocation lines with the information contained in a given set of density measures. The performance of the derived equations is benchmarked against other models proposed in the literature, using discrete dislocation dynamics simulations as a reference. As a benchmark problem we study dislocations moving in a highly heterogeneous, persistent-slip-band like geometry. We demonstrate that excellent agreement with discrete simulations can be obtained in terms of a very small number of averaged dislocation fields containing information about the edge and screw components of the total and excess (geometrically necessary) dislocation densities. From these the full dislocation orientation distribution which emerges as dislocations move through a channel-wall structure can be faithfully reconstructed.

  19. Calcitonin gene-related peptide regulation of glial cell-line derived neurotrophic factor in differentiated rat myotubes.

    Science.gov (United States)

    Rosa, Elyse; Cha, Jieun; Bain, James R; Fahnestock, Margaret

    2015-03-01

    Glial cell-line derived neurotrophic factor (GDNF) is the most potent trophic factor for motoneuron survival and neuromuscular junction formation. GDNF is upregulated in injured or denervated skeletal muscle and returns to normal levels following reinnervation. However, the mechanism by which GDNF is regulated in denervated muscle is not well understood. The nerve-derived neurotransmitter calcitonin gene-related peptide (CGRP) is upregulated following neuromuscular injury and is subsequently released from motoneurons at the neuromuscular junction. CGRP also promotes nerve regeneration, but the mechanism is not well understood. The current study investigates whether this increase in CGRP regulates GDNF, thus playing a key role in promoting regeneration of injured nerves. This study demonstrates that CGRP increases GDNF secretion without affecting its transcription or translation. Rat L6 myoblasts were differentiated into myotubes and subsequently treated with CGRP. GDNF mRNA expression levels were quantified by quantitative real-time reverse transcription-polymerase chain reaction, and secreted GDNF was quantified in the conditioned medium by ELISA. CGRP treatment increased secreted GDNF protein without altering GDNF mRNA levels. The translational inhibitor cycloheximide did not affect CGRP-induced GDNF secreted protein levels, whereas the secretional inhibitor brefeldin A blocked the CGRP-induced increase in GDNF. This study highlights the importance of injury-induced upregulation of CGRP by exposing its ability to increase GDNF levels and demonstrates a secretional mechanism for regulation of this key regeneration-promoting neurotrophic factor.

  20. Glial cell line-derived neurotrophic factor (GDNF) enhances sympathetic neurite growth in rat hearts at early developmental stages.

    Science.gov (United States)

    Miwa, Keiko; Lee, Jong-Kook; Takagishi, Yoshiko; Opthof, Tobias; Fu, Xianming; Kodama, Itsuo

    2010-12-01

    Molecular signaling of sympathetic innervation of myocardium is an unresolved issue. The purpose of this study was to investigate the effect of neurotrophic factors on sympathetic neurite growth towards cardiomyocytes. Cardiomyocytes (CMs) and sympathetic neurons (SNs) were isolated from neonatal rat hearts and superior cervical ganglia, and were co-cultured, either in a random or localized way. Neurite growth from SNs toward CMs was assessed by immunohistochemistry for neurofilament M and α-actinin in response to neurotrophic factors-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and a chemical repellent, semaphorin 3A. As a result, GDNF as well as NGF and BDNF stimulated neurite growth. GDNF enhanced neurite outgrowth even under the NGF-depleted culture condition, excluding an indirect effect of GDNF via NGF. Quantification of mRNA and protein by real-time PCR and immunohistochemistry at different developmental stages revealed that GDNF is abundantly expressed in the hearts of embryos and neonates, but not in adult hearts. GDNF plays an important role in inducing cardiac sympathetic innervation at the early developmental stages. A possible role in (re)innervation of injured or transplanted or cultured and transplanted myocardium may deserve investigation.

  1. Quantitative trait loci mapping for leaf length and leaf width in rice cv. IR64 derived lines.

    Science.gov (United States)

    Farooq, Muhammad; Tagle, Analiza G; Santos, Rizza E; Ebron, Leodegario A; Fujita, Daisuke; Kobayashi, Nobuya

    2010-06-01

    The present study was conducted to identify quantitative trait loci (QTLs) for leaf size traits in IR64 introgression lines (INLs). For this purpose, selected F(2) populations derived from crosses between recurrent parent IR64 and its derived INLs, unique for leaf length and leaf width, were used to confirm QTLs. A total of eight QTLs, mapped on three chromosomes, were identified for the four leaf size traits in six F(2) populations. A QTL for leaf length, qLLnpt-1, in HKL69 was identified around simple sequence repeat (SSR) marker RM3709 on chromosome 1. Two QTLs for flag leaf length, qFLLnpt-2 and qFLLnpt-4, in HFG39 were indentified on chromosomes 2 and 4, respectively. For flag leaf width, a QTL, qFLWnpt-4, in HFG39 was identified around RM17483 on chromosome 4. While another QTL for flag leaf width, qFLWnpt-1, in HFG27 was identified around RM3252 on chromosome 1. A QTL for leaf width, qLWnpt-2, in HKL75 was identified around RM7451 on chromosome 2. For leaf width, two QTLs, qLWnpt-4a, qLWnpt-4b, in HKL48 and HKL99 were identified around RM7208 and RM6909, respectively on chromosome 4. Results from this study suggest the possibilities to use marker-assisted selection and pyramiding these QTLs to improve rice water productivity.

  2. Frontal dermoid cyst coexisting with suprasellar craniopharyngioma: a spectrum of ectodermally derived epithelial-lined cystic lesions?

    Science.gov (United States)

    Abou-Al-Shaar, Hussam; Abd-El-Barr, Muhammad M; Zaidi, Hasan A; Russell-Goldman, Eleanor; Folkerth, Rebecca D; Laws, Edward R; Antonio Chiocca, E

    2016-12-01

    There is a wide group of lesions that may exist in the sellar and suprasellar regions. Embryologically, there is varying evidence that many of these entities may in fact represent a continuum of pathology deriving from a common ectodermal origin. The authors report a case of a concomitant suprasellar craniopharyngioma invading the third ventricle with a concurrent frontal lobe cystic dermoid tumor. A 21-year-old man presented to the authors' service with a 3-day history of worsening headache, nausea, vomiting, and blurry vision. Magnetic resonance imaging depicted a right frontal lobe lesion associated with a separate suprasellar cystic lesion invading the third ventricle. The patient underwent a right pterional craniotomy for resection of both lesions. Gross-total resection of the right frontal lesion was achieved, and subtotal resection of the suprasellar lesion was accomplished with some residual tumor adherent to the walls of the third ventricle. Histopathological examination of the resected right frontal lesion documented a diagnosis of dermoid cyst and, for the suprasellar lesion, a diagnosis of adamantinomatous craniopharyngioma. The occurrence of craniopharyngioma with dermoid cyst has not been reported in the literature before. Such an association might indeed suggest the previously reported hypothesis that these lesions represent a spectrum of ectodermally derived epithelial-lined cystic lesions.

  3. Urea derivates of ursolic, oleanolic and maslinic acid induce apoptosis and are selective cytotoxic for several human tumor cell lines.

    Science.gov (United States)

    Sommerwerk, Sven; Heller, Lucie; Kuhfs, Julia; Csuk, René

    2016-08-25

    2,3-Di-O-acetyl-maslinic acid benzylamide (5) has previously been shown to possess high cytotoxicity for a variety of human tumor cell lines while being of low cytotoxicity to non-malignant cells. Structural modifications performed on 5 revealed that the presence of these acetyl groups in 5 and the presence of (2β,3β)-configurated centers seems necessary for obtaining high cytotoxicity combined with best selectivity between malignant cells and non-malignant mouse fibroblasts. Compounds carrying an ursane skeleton showed weaker cytotoxicity than their oleanane derived analogs. In addition, the benzylamide function in compound 5 should be replaced by a phenylurea moiety to gain better cytotoxicity while retaining and improving the selectivity. Thus, maslinic acid derived N-[2β,3β-di-O-acetyl-17β-amino-28-norolean-12-en-17-yl]phenylurea (45) gave best results showing EC50 = 0.9 μM (for A2780 ovarian cancer cells) with EC50 > 120 μM for fibroblasts (NIH 3T3) and triggered apoptosis while caspase-3 was not activated by this compound.

  4. Effective and Steady Differentiation of a Clonal Derivative of P19CL6 Embryonal Carcinoma Cell Line into Beating Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Itsuki Mueller

    2010-01-01

    Full Text Available The P19CL6 cell line is a useful model to study cardiac differentiation in vitro. However, large variations were noticed in the differentiation rates among previous reports as well as our individual experiments. To overcome the unstable differentiation, we established P19CL6-A1, a new clonal derivative of P19CL6 that could differentiate into cardiomyocytes more efficiently and stably than the parent using the double stimulation with 5-Aza and DMSO based on the previous report. We also introduced a new software, Visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms. Using P19CL6-A1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents. These indicate that a combination of P19CL6-A1 and Visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing.

  5. Photophysical Properties of Pheophorbide-a Derivatives and Their Photodynamic Therapeutic Effects on a Tumor Cell Line In Vitro

    Directory of Open Access Journals (Sweden)

    Kang-Kyun Wang

    2014-01-01

    Full Text Available Pheophorbide-a derivatives have been reported to be potential photosensitizers for photodynamic therapy (PDT. In this study, photophysics of pheophorbide-a derivatives (PaDs were investigated along with their photodynamic tumoricidal effect in vitro. PaDs were modified by changing the coil length and/or making the hydroxyl group (–OH substitutions. Their photophysical properties were studied by steady-state and time-resolved spectroscopic methods. The photodynamic tumoricidal effect was evaluated in the mouse breast cancer cell line (EMT6. Lifetime and quantum yield of fluorescence and quantum yields of triplet state and singlet oxygen were studied to determine the dynamic energy flow. The coil length of the substituted alkyl group did not significantly affect the spectroscopic properties. However, the substitution with the hydroxyl group increased the quantum yields of the triplet state and the singlet oxygen due to the enhanced intersystem crossing. In order to check the application possibility as a photodynamic therapy agent, the PaDs with hydroxyl group were studied for the cellular affinity and the photodynamic tumoricidal effect of EMT6. The results showed that the cellular affinity and the photodynamic tumoricidal effect of PaDs with the hydroxyl group depended on the coil-length of the substituted alkyl group.

  6. New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

    Science.gov (United States)

    Saraiva-Pava, Kathy; Navabi, Nazanin; Skoog, Emma C; Lindén, Sara K; Oleastro, Mónica; Roxo-Rosa, Mónica

    2015-01-01

    AIM: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection. METHODS: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones’ adherence properties, expression of cell-cell junctions’ markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence. RESULTS: Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Lex), Ley and Lea and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans. Entailing good gastric properties, both NCI-hTERT-clones were found to produce

  7. A thermoreversible polymer mediates controlled release of glial cell line-derived neurotrophic factor to enhance kidney regeneration.

    Science.gov (United States)

    Gheisari, Yousof; Yokoo, Takashi; Matsumoto, Kei; Fukui, Akira; Sugimoto, Naomi; Ohashi, Toya; Kawamura, Tetsuya; Hosoya, Tatsuo; Kobayashi, Eiji

    2010-08-01

    Previously, we reported that human mesenchymal stem cells (hMSCs) that were cultivated in growing embryos differentiated in an appropriate developmental milieu, thereby facilitating the development of a functional renal unit. However, this approach required transfection with an adenovirus that expressed glial cell line-derived neurotrophic factor (GDNF) to enhance the development of hMSC-derived renal tissue, and safety issues restrict the clinical use of such viral vectors. To circumvent this problem, we tested an artificial polymer as a means to diffuse GDNF. This GDNF-polymer, which exists in liquid form at 4 degrees C but becomes a hydrogel upon heating to 37 degrees C, was used as a thermoreversible switch, allowing the injection of hMSCs at low viscosity using a mouth pipette, with subsequent slow diffusion of GDNF as it solidified. The polymer, which was dissolved in a solution of GDNF at 4 degrees C and then maintained at 37 degrees C, acted as a diffuser of GDNF for more than 48 h. LacZ-transfected hMSCs and the GDNF-polymer (at 4 degrees C) were placed in the nephrogenic sites of growing rat embryos that were maintained at 37 degrees C. Forty-eight hours later, the resultant kidney anlagen were dissected out and allowed to continue developing for 6 days in vitro. Whole-organ X-Gal staining and fluorescence activated cell sorter analysis showed that the number of hMSC-derived cells was significantly increased in developed anlagen that have been generated from hMSCs plus GDNF-polymer compared with those from hMSCs plus GDNF-containing medium and was comparable to those from adenovirus-transfected hMSCs. These findings suggest that the GDNF-polymer can be used as a diffuser of GDNF for kidney organogenesis.

  8. Cytotoxicity of nano-hydroxyapatite on human-derived oral epithelium cell line: an in vitro study

    Directory of Open Access Journals (Sweden)

    Farid Abassi

    2016-08-01

    Full Text Available Background: Hydroxyapatite nanoparticles have a more surface contact and solubility than conventional hydroxyapatite. Hydroxynanoparticles enhances the biological and mechanical properties of new regenerated tissues. The hydroxyapatite nanoparticles have received attention as a new and effective osseous graft for using as scaffolds in bone regeneration. The reports on hydroxyapatite nanoparticles biocompatibility are controversial. It has been shown that hydroxyapatite nanoparticles induces inflammatory reaction and apoptosis. The aim of the present study was to evaluate the cytotoxicity of nano-hydroxyapatite on the human epithelial cells. Methods: The study was experimental and completed in vitro. The study was carried out in department of Immonulogy, Faculty of Medicine, Shahid Beheshti University of Medical Sciences in November 2014. The human-derived oral epithelium cell line (KB obtained from Pasteur Institute, Tehran, Iran were exposed to hydroxyapatite nanoparticles at 0.01, 0.05, 0.1, 0.5, 0.75, 1, 2.5 and 5 mg/ml concentrations in 24, 48 and 72 hours. Rod-shaped hydroxyapatite nanoparticles with 99% purity and maximum 100 nm sized particles were used. Methylthiazol tetrazolium bromide (MTT method was employed for cell vitality evaluation. Enzyme-linked immunosorbent assay (ELISA was used for assessing the viability of cells. Distilled water and fetal bovine serum (FBS were positive and negative controls. ANOVA and Duncan tests were used for statistical analysis. Results: The cytotoxicity of different concentrations of hydroxyapatite nanoparticles on human-derived oral epithelium cell line in 24 (P< 0.001, 48 (P< 0.001 and 72 hours (P< 0.001 was significantly different. The nano-hydroxyapatite particles at 0.5 to 1 mg/ml had the highest cytotoxicity effect on human-derived oral epithelium cells in 24, 48 and 72 hours. Lower concentrations than 0.05 mg/ml had the best biocompatibility properties in 24, 48 and 72 hours. Conclusion

  9. 3. Chromosomal instability in B-lymphoblasotoid cell lines from Werner's and Bloom's syndrome patients

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Werner's syndrome (WS) and Bloom's syndrome (BS) are rare autosomal recessive diseases in which the feature of premature aging and the elevated risk of neoplasia may be associated with genomic instability. To cha-racterize the genomic instability of WS and BS, B-lymphoblastoid cell lines (LCLs) from WS and BS patients were cytogenetically analyzed, comparing to those from healthy donors. Although all

  10. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line.

    Science.gov (United States)

    Toyohara, Yukiyo; Hashitani, Susumu; Kishimoto, Hiromitsu; Noguchi, Kazuma; Yamamoto, Nobuto; Urade, Masahiro

    2011-07-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model.

  11. Forward subtractive libraries containing genes transactivated by dexamethasone in ataxia-telangiectasia lymphoblastoid cells.

    Science.gov (United States)

    Biagiotti, Sara; Menotta, Michele; Giacomini, Elisa; Radici, Lucia; Bianchi, Marzia; Bozzao, Cristina; Chessa, Luciana; Magnani, Mauro

    2014-07-01

    Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder caused by biallelic mutations in the Ataxia Telangiectasia-mutated gene. A-T shows a complex phenotype ranging from early-onset progressive neurodegeneration to immunodeficiencies, high incidence of infections, and tumors. Unfortunately, no therapy is up to now available for treating this condition. Recently, the short term treatment of ataxia-telangiectasia patients with glucocorticoids was shown to improve their neurological symptoms and possibly reverse cerebellar atrophy. Thus, corticosteroids represent an attractive approach for the treatment of this neurodegenerative disease. However, the molecular mechanism involved in glucocorticoid action in A-T is yet unknown. The aim of our work is to construct cDNA libraries containing those genes which are transactivated by the glucocorticoid analogue, dexamethasone, in A-T human cells. For this purpose, suppression subtractive hybridization has been performed on ATM-null lymphoblastoid cell transcriptome extracted following drug administration. Annotation of whole genes contained in the libraries has been obtained by coupling subtractive hybridization with microarray analysis. Positive transcripts have been validated by quantitative PCR. Through in silico analyses, identified genes have been classified on the basis of the pathway in which they are involved, being able to address signaling required for dexamethasone action. Most of the induced transcripts are involved in metabolic processes and regulation of cellular processes. Our results can help to unravel the mechanism of glucocorticoid action in the reversion of A-T phenotype. Moreover, the induction of a specific region of the ATM transcript has been identified as putative biomarker predictive of dexamethasone efficacy on ataxic patients.

  12. A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells.

    Science.gov (United States)

    Kimura, Aoi; Miyata, Atsuro; Honma, Masamitsu

    2013-09-01

    The comet assay has been widely used as a genotoxicity test for detecting primary DNA damage in individual cells. The micronucleus (MN) test is also a well-established assay for detecting clastogenicity and aneugenicity. A combination of the comet assay (COM) and MN test is capable of detecting a variety of genotoxic potentials as an in vitro screening system. Although the in vitro MN test has a robust protocol and Organisation for Economic Co-operation and Development (OECD) test guideline, the in vitro COM does not. To establish a robust protocol for the COM and to compare its sensitivity with that of the MN, we conducted COM and MN concurrently for five genotoxic agents (ethyl methanesulfonate, methyl methanesulfonate, hydrogen peroxide, gamma-rays and mitomycin C) and one non-genotoxic agent (triton X-100), using human lymphoblastoid TK6 cells. Relative cell count (RCC), relative population doubling (RPD), relative increase in cell count (RICC) and relative cell viability determined by trypan blue dye-exclusion assay (TBDE) were employed as cytotoxic measurements. However, the relative cell viability determined by TBDE just after the treatment was not an appropriate parameter of cytotoxicity for the genotoxic agents because it remained constant even at the highest doses, which showed severe cytotoxicity by RCC, RPD and RICC. The results of the COM showed qualitative agreement (positive or negative) with those of the MN except for mitomycin C, which is an interstrand cross-linker. The COM always required higher doses than the MN to detect the genotoxic potential of the genotoxic agents under the test conditions applied here. The doses that induced a comet tail always yielded test guideline for MN because of their high cytotoxicity. These results are helpful for interpreting the results of the COM and MN in in vitro genotoxic hazard assessments. Further investigation is required to standardise the COM.

  13. Understanding the role of keratins 8 and 18 in neoplastic potential of breast cancer derived cell lines.

    Directory of Open Access Journals (Sweden)

    Sapna V Iyer

    Full Text Available BACKGROUND: Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER, progesterone receptor (PGR and epidermal growth factor receptor 2 (HER2 in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays. CONCLUSIONS/SIGNIFICANCE: These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.

  14. Associations of corticosterone and testosterone with alcohol drinking in F2 populations derived from AA and ANA rat lines.

    Science.gov (United States)

    Etelälahti, Tiina J; Saarikoski, Sirkku T; Eriksson, C J Peter

    2011-08-01

    In our previous studies on alcohol-preferring AA (Alko alcohol) and nonpreferring ANA (Alko nonalcohol) rats, we have observed that the AA rats exhibit lower endogenous levels of corticosterone, higher testosterone levels, and more frequent alcohol-induced testosterone elevations when compared with ANA rats. The objective of the present study was to get more conclusive evidence for the potential role of the hypothalamus-pituitary-adrenal and hypothalamus-pituitary-gonadal axes in alcohol drinking by using the F2 experimental design. Alcohol-preferring AA and alcohol-nonpreferring ANA rat lines were crossbred to form a F1 population from which the final F2 population was derived. Male animals were challenged with a priming alcohol dose after which a 3 weeks' voluntary alcohol drinking period took place. After a washout period of 1 week, one-half of the 40 highest and 40 lowest alcohol drinkers were challenged with a second dose of alcohol and the other half with saline. Serum testosterone and corticosterone levels were measured before and during the test. Higher endogenous testosterone levels were detected in the rats of the high alcohol consumption group compared with the low consumption group. Also supporting the original AA/ANA line differences, a trend for lower endogenous corticosterone levels were measured in the high alcohol consumption group compared with the low consumption group. The alcohol challenge test after the drinking period resulted in a higher frequency (38%) of testosterone elevations in the high drinkers compared with the low drinkers (5%). The present data confirms the validity of the positive connections between testosterone elevation and increased alcohol drinking, as well as between testosterone reduction and decreased alcohol drinking, in AA and ANA rats.

  15. Expression of brain-derived neurotrophic factor and TrkB in the lateral line system of zebrafish during development.

    Science.gov (United States)

    Germanà, A; Laurà, R; Montalbano, G; Guerrera, M C; Amato, V; Zichichi, R; Campo, S; Ciriaco, E; Vega, J A

    2010-07-01

    The neuromasts of the lateral line system are regarded as a model to study the mechanisms of hearing, deafness, and ototoxicity. The neurotrophins (NTs), especially brain-derived neurotrophic factor (BDNF), and its signaling receptor TrkB are involved in the development and maintenance of neuromasts. To know the period in which the BDNF/TrkB complex has more effects in the neuromast biology, the age-related changes were studied. Normal zebrafish from 10 to 180 days post-fertilization (dpf), as well as transgenic ET4 zebrafish 10 and 20 dpf, was analyzed using qRT-PCR, western blot, and immunohistochemistry. BDNF and TrkB mRNAs followed a parallel course, peaking at 20 dpf, and thereafter progressively decreased. Specific immunoreactivity for BDNF and TrkB was found co-localized in all hairy cells of neuromasts in 20 and 30 dpf; then, the number of immunoreactive cells decreased, and by 180 dpf BDNF remains restricted to a subpopulation of hairy cells, and TrkB to a few number of sensory and non-sensory cells. At all ages examined, TrkB immunoreactivity was detected in sensory ganglia innervating the neuromasts. The present results demonstrate that there is a parallel time-related decline in the expression of BDNF and TrkB in zebrafish. Also, the patterns of cell expression suggest that autocrine/paracrine mechanisms for this NT system might occur within the neuromasts. Because TrkB in lateral line ganglia did not vary with age, their neurons are potentially capable to respond to BDNF during the entire lifespan of zebrafish.

  16. Glial cell line-derived neurotrophic factor promotes barrier maturation and wound healing in intestinal epithelial cells in vitro.

    Science.gov (United States)

    Meir, Michael; Flemming, Sven; Burkard, Natalie; Bergauer, Lisa; Metzger, Marco; Germer, Christoph-Thomas; Schlegel, Nicolas

    2015-10-15

    Recent data suggest that neurotrophic factors from the enteric nervous system are involved in intestinal epithelial barrier regulation. In this context the glial cell line-derived neurotrophic factor (GDNF) was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease (IBD). We further investigated the potential role and mechanisms of GDNF in the regulation of intestinal barrier functions. Immunostaining of human gut specimen showed positive GDNF staining in enteric neuronal plexus and in enterocytes. In Western blots of the intestinal epithelial cell lines Caco2 and HT29B6, significant amounts of GDNF were detected, suggesting that enterocytes represent an additional source of GDNF. Application of recombinant GDNF on Caco2 and HT29B6 cells for 24 h resulted in significant epithelial barrier stabilization in monolayers with immature barrier functions. Wound-healing assays showed a significantly faster closure of the wounded areas after GDNF application. GDNF augmented cAMP levels and led to significant inactivation of p38 MAPK in immature cells. Activation of p38 MAPK signaling by SB-202190 mimicked GDNF-induced barrier maturation, whereas the p38 MAPK activator anisomycin blocked GDNF-induced effects. Increasing cAMP levels had adverse effects on barrier maturation, as revealed by permeability measurements. However, increased cAMP augmented the proliferation rate in Caco2 cells, and GDNF-induced proliferation of epithelial cells was abrogated by the PKA inhibitor H89. Our data show that enterocytes represent an additional source of GDNF synthesis. GDNF contributes to wound healing in a cAMP/PKA-dependent manner and promotes barrier maturation in immature enterocytes cells by inactivation of p38 MAPK signaling.

  17. Human papillomavirus type 18 E6 and E7 genes integrate into human hepatoma derived cell line Hep G2.

    Science.gov (United States)

    Ma, Tianzhong; Su, Zhongjing; Chen, Ling; Liu, Shuyan; Zhu, Ningxia; Wen, Lifeng; Yuan, Yan; Lv, Leili; Chen, Xiancai; Huang, Jianmin; Chen, Haibin

    2012-01-01

    Human papillomaviruses have been linked causally to some human cancers such as cervical carcinoma, but there is very little research addressing the effect of HPV infection on human liver cells. We chose the human hepatoma derived cell line Hep G2 to investigate whether HPV gene integration took place in liver cells as well. We applied PCR to detect the possible integration of HPV genes in Hep G2 cells. We also investigated the expression of the integrated E6 and E7 genes by using RT-PCR and Western blotting. Then, we silenced E6 and E7 expression and checked the cell proliferation and apoptosis in Hep G2 cells. Furthermore, we analyzed the potential genes involved in cell cycle and apoptosis regulatory pathways. Finally, we used in situ hybridization to detect HPV 16/18 in hepatocellular carcinoma samples. Hep G2 cell line contains integrated HPV 18 DNA, leading to the expression of the E6 and E7 oncogenic proteins. Knockdown of the E7 and E6 genes expression reduced cell proliferation, caused the cell cycle arrest at the S phase, and increased apoptosis. The human cell cycle and apoptosis real-time PCR arrays analysis demonstrated E6 and E7-mediated regulation of some genes such as Cyclin H, UBA1, E2F4, p53, p107, FASLG, NOL3 and CASP14. HPV16/18 was found in only 9% (9/100) of patients with hepatocellular carcinoma. Our investigations showed that HPV 18 E6 and E7 genes can be integrated into the Hep G2, and we observed a low prevalence of HPV 16/18 in hepatocellular carcinoma samples. However, the precise risk of HPV as causative agent of hepatocellular carcinoma needs further study.

  18. Whole-blastocyst culture followed by laser drilling technology enhances the efficiency of inner cell mass isolation and embryonic stem cell derivation from good- and poor-quality mouse embryos: new insights for derivation of human embryonic stem cell lines.

    Science.gov (United States)

    Cortes, J L; Sánchez, L; Catalina, P; Cobo, F; Bueno, C; Martínez-Ramirez, A; Barroso, A; Cabrera, C; Ligero, G; Montes, R; Rubio, R; Nieto, A; Menendez, P

    2008-04-01

    The optimization of human embryonic stem (hES) cell line derivation methods is challenging because many worldwide laboratories have neither access to spare human embryos nor ethical approval for using supernumerary human embryos for hES cell derivation purposes. Additionally, studies performed directly on human embryos imply a waste of precious human biological material. In this study, we developed a new strategy based on the combination of whole-blastocyst culture followed by laser drilling destruction of the trophoectoderm for improving the efficiency of inner cell mass (ICM) isolation and ES cell derivation using murine embryos. Embryos were divided into good- and poor-quality embryos. We demonstrate that the efficiency of both ICM isolation and ES cell derivation using this strategy is significantly superior to whole-blastocyst culture or laser drilling technology itself. Regardless of the ICM isolation method, the ES cell establishment depends on a feeder cell growth surface. Importantly, this combined methodology can be successfully applied to poor-quality blastocysts that otherwise would not be suitable for laser drilling itself nor immunosurgery in an attempt to derive ES cell lines due to the inability to distinguish the ICM. The ES cell lines derived by this combined method were characterized and shown to maintain a typical morphology, undifferentiated phenotype, and in vitro and in vivo three germ layer differentiation potential. Finally, all ES cell lines established using either technology acquired an aneuploid karyotype after extended culture periods, suggesting that the method used for ES cell derivation does not seem to influence the karyotype of the ES cells after extended culture. This methodology may open up new avenues for further improvements for the derivation of hES cells, the majority of which are derived from frozen, poor-quality human embryos.

  19. To determine the cytotoxicity of chlorambucil and one of its nitro-derivatives, conjugated to prasterone and pregnenolone, towards eight human cancer cell-lines.

    Science.gov (United States)

    Shervington, Leroy A; Smith, Nigel; Norman, Emma; Ward, Timothy; Phillips, Roger; Shervington, Amal

    2009-07-01

    Four ester prodrugs derived from the bifunctional alkylating agent chlorambucil, and one of its nitro-derivatives, 3-nitrochlorambucil conjugated to prasterone and pregnenolone, were synthesized and tested for their cytotoxic activity against eight human cell lines, using the standard MTT assay. A comparison between the esters and the controls, namely chlorambucil and 3-nitrochlorambucil would suggest that all four esters possess to varying degrees, specificity towards the breast adenocarcinoma cell line (MDA-mb468) than the other seven cells' lines tested. The overall findings are encouraging since it infers that these lipophilic esters not only have the ability to traverse specific cell membranes but also exhibit cytotoxicity towards most of the cell lines tested.

  20. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

    Directory of Open Access Journals (Sweden)

    Jin Hee Kim

    Full Text Available When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i there are no publicly available cloning vectors harboring a 2A peptide gene and (ii comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

  1. Culture of mouse amniotic fluid-derived cells on irradiated STO feeders results in the generation of primitive endoderm cell lines capable of self-renewal in vitro.

    Science.gov (United States)

    Babic, Aleksandar M; Jang, Sunyoung; Nicolov, Eugenia; Voicu, Horatiu; Luckey, Chance J

    2013-01-01

    The cells present in amniotic fluid (AF) are currently used for prenatal diagnosis of fetal anomalies but are also a potential source of cells for cell therapy. To better characterize putative progenitor cell populations present in AF, we used culture conditions that support self-renewal to determine if these promoted the generation of stable cell lines from AF-derived cells (AFC). Cells isolated from E11.5 mouse were cultured on irradiated STO fibroblast feeder layers in human embryonic germ cell derivation conditions. The cultures grew multicellular epithelial colonies that could be repropagated from single cells. Reverse transcription semiquantitative polymerase chain reaction of established cell lines revealed that they belonged to the extraembryonic endoderm (ExEn) expressing high levels of Gata6, Gata4, Sox17, Foxa2 and Sox7 mRNA. Hierarchical clustering based on the whole transcriptome expression profile of the AFC lines (AFCL) shows significant correlation between transcription profiles of AFCL and blastocyst-derived XEN, an ExEn cell line. In vitro differentiation of AFCL results in the generation of cells expressing albumin and α-fetoprotein (AFP), while intramuscular injection of AFCL into immunodeficient mice produced AFP-positive tumors with primitive endodermal appearance. Hence, E11.5 mouse AF contains cells that efficiently produce XEN lines. These AF-derived XEN lines do not spontaneously differentiate into embryonic-type cells but are phenotypically stable and have the capacity for extensive expansion. The lack of requirement for reprogramming factors to turn AF-derived progenitor cells into stable cell lines capable of massive expansion together with the known ability of ExEn to contribute to embryonic tissue suggests that this cell type may be a candidate for banking for cell therapies. © 2013 S. Karger AG, Basel

  2. International Conference on Harmonisation; guidance on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin; availability--FDA. Notice.

    Science.gov (United States)

    1998-09-24

    The Food and Drug Administration (FDA) is publishing a guidance entitled "Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin." The guidance was prepared under the auspices of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The guidance describes the testing and evaluation of the viral safety of biotechnology products derived from characterized cell lines of human or animal origin, and outlines data that should be submitted in marketing applications.

  3. Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor.

    Science.gov (United States)

    Liu, Zhong-wei; Li, Wen-qiang; Wang, Jun-kui; Ma, Xian-cang; Liang, Chen; Liu, Peng; Chu, Zheng; Dang, Yong-hui

    2014-12-01

    This study investigated the potential application of a zirconium oxide (ZrO2) ceramic foam culturing system to the production of glial cell line-derived neurotrophic factor (GDNF). Three sets of ZrO2 ceramic foams with different pore densities of 10, 20, and 30 pores per linear inch (PPI) were prepared to support a 3D culturing system. After primary astrocytes were cultured in these systems, production yields of GDNF were evaluated. The biomaterial biocompatibility, cell proliferation and activation of cellular signaling pathways in GDNF synthesis and secretion in the culturing systems were also assessed and compared with a conventional culturing system. In this study, we found that the ZrO2 ceramic foam culturing system was biocompatible, using which the GDNF yields were elevated and sustained by stimulated cell proliferation and activation of signaling pathways in astrocytes cultured in the system. In conclusion, the ZrO2 ceramic foam is promising for the development of a GDNF mass production device for Parkinson's disease treatment.

  4. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  5. The effect of line damping, magneto-optics and parasitic light on the derivation of sunspot vector magnetic fields

    Science.gov (United States)

    Skumanich, A.; Lites, B. W.

    1985-01-01

    The least square fitting of Stokes observations of sunspots using a Milne-Eddington-Unno model appears to lead, in many circumstances, to various inconsistencies such as anomalously large doppler widths and, hence, small magnetic fields which are significantly below those inferred solely from the Zeeman splitting in the intensity profile. It is found that the introduction of additional physics into the model such as the inclusion of damping wings and magneto-optic birefrigence significantly improves the fit to Stokes parameters. Model fits excluding the intensity profile, i.e., of both magnitude as well as spectral shape of the polarization parameters alone, suggest that parasitic light in the intensity profile may also be a source of inconsistencies. The consequences of the physical changes on the vector properties of the field derived from the Fe I lambda 6173 line for the 17 November 1975 spot as well as on the thermodynamic state are discussed. A Doppler width delta lambda (D) - 25mA is bound to be consistent with a low spot temperature and microturbulence, and a damping constant of a = 0.2.

  6. Decreased glial cell line-derived neurotrophic factor levels in patients with depression: a meta-analytic study.

    Science.gov (United States)

    Lin, Pao-Yen; Tseng, Ping-Tao

    2015-04-01

    Glial cell-line derived neurotrophic factor (GDNF) has been shown to promote development, differentiation, and protection of CNS neurons and was thought to play an important role in various neuropsychiatric disorders. Several studies have examined the GDNF levels in patients with depression but shown inconsistent results. In this study, we compared blood GDNF levels between depressive patients and control subjects through meta-analytic method. The effect sizes (ESs) from all eligible studies were synthesized by using a random effect model. In this meta-analysis, we included 526 patients and 502 control subjects from 12 original articles. Compared to control subjects, blood GDNF levels are significantly decreased in patients with depression (ES = -0.62, p = 0.0011). However, significant heterogeneity was found among included studies. Through subgroup analysis, we found that GDNF was still decreased in studies with major depressive disorder (ES = -0.73, p = 0.0001); in studies with non-old-age depression (ES = -1.25, p = 0.0001), but not with old-age depression; and in studies using serum samples (ES = -0.86, p GDNF levels as a biomarker of depression as a whole, but the results were modulated by psychiatric diagnosis, age of included subjects, and sampling sources. With these results, future studies are required to examine whether effective antidepressant treatment is associated with an increase in serum GDNF levels.

  7. Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells.

    Science.gov (United States)

    Vianney, John-Mary; Miller, Damon A; Spitsbergen, John M

    2014-11-01

    Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle.

  8. Sympathetic Innervation Induced in Engrafted Engineered Cardiomyocyte Sheets by Glial Cell Line Derived Neurotrophic Factor In Vivo

    Directory of Open Access Journals (Sweden)

    Xian-ming Fu

    2013-01-01

    Full Text Available The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF. GFP-transgene (control group or GDNF overexpressing (GDNF group engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF.

  9. Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Ruttachuk Rungsiwiwut

    2016-01-01

    Full Text Available Although human pluripotent stem cells (hPSCs can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS prior to coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFβ1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.

  10. Quantitative trait loci for rice yield-related traits using recombinant inbred lines derived from two diverse cultivars

    Indian Academy of Sciences (India)

    Xu Feng Bai; Li Jun Luo; Wen Hao Yan; Mallikarjuna Rao Kovi; Yong Zhong Xing

    2011-08-01

    The thousand-grain weight and spikelets per panicle directly contribute to rice yield. Heading date and plant height also greatly influence the yield. Dissection of genetic bases of yield-related traits would provide tools for yield improvement. In this study, quantitative trait loci (QTL) mapping for spikelets per panicle, thousand-grain weight, heading date and plant height was performed using recombinant inbred lines derived from a cross between two diverse cultivars, Nanyangzhan and Chuan7. In total, 20 QTLs were identified for four traits. They were located to 11 chromosomes except on chromosome 4. Seven and five QTLs were detected for thousand-grain weight and spikelets per panicle, respectively. Four QTLs were identified for both heading date and plant height. About half the QTLs were commonly detected in both years, 2006 and 2007. Six QTLs are being reported for the first time. Two QTL clusters were identified in regions flanked by RM22065 and RM5720 on chromosome 7 and by RM502 and RM264 on chromosome 8, respectively. The parent, Nanyangzhan with heavy thousand-grain weight, carried alleles with increased effects on all seven thousand-grain weight QTL, which explained why there was no transgressive segregation for thousand-grain weight in the population. In contrast, Chuan7 with more spikelets per panicle carried positive alleles at all five spikelets per panicle QTL except qspp5. Further work on distinction between pleiotropic QTL and linked QTL is needed in two yield-related QTL clusters.

  11. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

    Science.gov (United States)

    Charoenviriyakul, Chonlada; Takahashi, Yuki; Morishita, Masaki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2017-01-01

    Exosomes are small membrane vesicles secreted from cells and are expected to be used as drug delivery systems. Important characteristics of exosomes, such as yield, physicochemical properties, and pharmacokinetics, may be different among different cell types. However, there is limited information about the effect of cell type on these characteristics. In the present study, we evaluated these characteristics of exosomes derived from five different types of mouse cell lines: B16BL6 murine melanoma cells, C2C12 murine myoblast cells, NIH3T3 murine fibroblasts cells, MAEC murine aortic endothelial cells, and RAW264.7 murine macrophage-like cells. Exosomes were collected using a differential ultracentrifugation method. The exosomes collected from all the cell types were negatively charged globular vesicles with a diameter of approximately 100nm. C2C12 and RAW264.7 cells produced more exosomes than the other types of cells. The exosomes were labeled with a fusion protein of Gaussia luciferase and lactadherin to evaluate their pharmacokinetics. After intravenous injection into mice, all the exosomes rapidly disappeared from the systemic circulation and mainly distributed to the liver. In conclusion, the exosome yield was significantly different among the cell types, and all the exosomes evaluated in this study showed comparable physicochemical and pharmacokinetic properties.

  12. Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line.

    Science.gov (United States)

    Liu, Chuan; Duan, Weixia; Xu, Shangcheng; Chen, Chunhai; He, Mindi; Zhang, Lei; Yu, Zhengping; Zhou, Zhou

    2013-03-27

    Whether exposure to radiofrequency electromagnetic radiation (RF-EMR) emitted from mobile phones can induce DNA damage in male germ cells remains unclear. In this study, we conducted a 24h intermittent exposure (5 min on and 10 min off) of a mouse spermatocyte-derived GC-2 cell line to 1800 MHz Global System for Mobile Communication (GSM) signals in GSM-Talk mode at specific absorption rates (SAR) of 1 W/kg, 2 W/kg or 4 W/kg. Subsequently, through the use of formamidopyrimidine DNA glycosylase (FPG) in a modified comet assay, we determined that the extent of DNA migration was significantly increased at a SAR of 4 W/kg. Flow cytometry analysis demonstrated that levels of the DNA adduct 8-oxoguanine (8-oxoG) were also increased at a SAR of 4 W/kg. These increases were concomitant with similar increases in the generation of reactive oxygen species (ROS); these phenomena were mitigated by co-treatment with the antioxidant α-tocopherol. However, no detectable DNA strand breakage was observed by the alkaline comet assay. Taking together, these findings may imply the novel possibility that RF-EMR with insufficient energy for the direct induction of DNA strand breaks may produce genotoxicity through oxidative DNA base damage in male germ cells.

  13. Alien DNA introgression and wheat DNA rearrangements in a stable wheat line derived from the early generation of distant hybridization

    Institute of Scientific and Technical Information of China (English)

    ZHANG Lianquan; LIU Dengcai; YAN Zehong; ZHENG Youliang

    2005-01-01

    Polyploidy has been found to be common in plants. Bread or common wheat (Triticum aestivum L., 2n = 42) is a good example of allopolyploid made up of three diploid genomes A, B and D. In recent years, by the study of mimicking the origination of common wheat, it was found that changes of DNA sequence and gene expression occurred at the early stages of artificial allohexaploid between tetraploid wheat and Aegilops tauschii, which was probably favorable to genetic diploidization of new synthetic hexaploid wheat. Common wheat 99L2 is a new line stable in genetic, which was derived from the early self-pollinated generation of wide hybrids between common wheat and rye. In this study, it was found that at least two rye DNA segments had been introgressed into 99L2. This result suggested that a mechanism of alien DNA introgression may exist, which was different from the traditional mechanism of chromosome pairing and DNA recombination between wheat and alien species. Meanwhile, during the introgression process of alien rye DNA segments, the changes in DNA sequences of wheat itself occurred.

  14. Synthesis and Evaluation of New Pyrazoline Derivatives as Potential Anticancer Agents in HepG-2 Cell Line

    Directory of Open Access Journals (Sweden)

    Weijie Xu

    2017-03-01

    Full Text Available Cancer is a major public health concern worldwide. Adverse effects of cancer treatments still compromise patients’ quality of life. To identify new potential anticancer agents, a series of novel pyrazoline derivatives were synthesized and evaluated for cytotoxic effects on HepG-2 (human liver hepatocellular carcinoma cell line and primary hepatocytes. Compound structures were confirmed by 1H-NMR, mass spectrometry, and infrared imaging. An in vitro assay demonstrated that several compounds exerted cytotoxicity in the micromolar range. Benzo[b]thiophen-2-yl-[5-(4-hydroxy-3,5-dimethoxy-phenyl-3-(2-hydroxy-phenyl-4,5-dihydo-pyrazol-1-yl]-methanone (b17 was the most effective anticancer agent against HepG-2 cells owing to its notable inhibitory effect on HepG-2 with an IC50 value of 3.57 µM when compared with cisplatin (IC50 = 8.45 µM and low cytotoxicity against primary hepatocytes. Cell cycle analysis and apoptosis/necrosis evaluation using this compound revealed that b17 notably arrested HepG-2 cells in the G2/M phase and induced HepG-2 cells apoptosis. Our findings indicate that compound b17 may be a promising anticancer drug candidate.

  15. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    Science.gov (United States)

    Lin, Chih-Lang; Chien, Rong-Nan; Lin, Shi-Ming; Ke, Po-Yuan; Lin, Chen-Chun; Yeh, Chau-Ting

    2013-01-01

    Occult hepatitis B virus (HBV) infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg). Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV) and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2). Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  16. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    Directory of Open Access Journals (Sweden)

    Chih-Lang Lin

    Full Text Available Occult hepatitis B virus (HBV infection is defined as persistence of HBV DNA in liver tissues, with or without detectability of HBV DNA in the serum, in individuals with negative serum HBV surface antigen (HBsAg. Despite accumulating evidence suggesting its important clinical roles, the molecular and virological basis of occult hepatitis B remains unclear. In an attempt to establish new hepatoma cell lines, we achieved a new cell line derived from a hepatoma patient with chronic hepatitis C virus (HCV and occult HBV infection. Characterization of this cell line revealed previously unrecognized properties. Two novel human hepatoma cell lines were established. Hep-Y1 was derived from a male hepatoma patient negative for HCV and HBV infection. Hep-Y2 was derived from a female hepatoma patient suffering from chronic HCV and occult HBV infection. Morphological, cytogenetic and functional studies were performed. Permissiveness to HBV infection was assessed. Both cell lines showed typical hepatocyte-like morphology under phase-contrast and electron microscopy and expressed alpha-fetoprotein, albumin, transferrin, and aldolase B. Cytogenetic analysis revealed extensive chromosomal anomalies. An extrachromosomal form of HBV DNA persisted in the nuclear fraction of Hep-Y2 cells, while no HBsAg was detected in the medium. After treated with 2% dimethyl sulfoxide, both cell lines were permissive for exogenous HBV infection with transient elevation of the replication intermediates in the cytosol with detectable viral antigens by immunoflurescence analysis. In conclusions, we established two new hepatoma cell lines including one from occult HBV infection (Hep-Y2. Both cell lines were permissive for HBV infection. Additionally, Hep-Y2 cells carried persistent extrachromosomal HBV DNA in the nuclei. This cell line could serve as a useful tool to establish the molecular and virological basis of occult HBV infection.

  17. Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations

    DEFF Research Database (Denmark)

    Momcilovic, Olga; Sivapatham, Renuka; Oron, Tal Ronnen

    2016-01-01

    We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight...... of these lines (one of each SNCA, LRRK2 and GBA, four PARK2 lines, and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line...... to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation, no mutation-specific clustering or difference was observed, though consistent changes in patient lines were detected in genes associated mitochondrial biology...

  18. Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19

    Science.gov (United States)

    Totipotent embryonic stem cell lines have not been established from ungulates, however, we have developed several somatic cell lines from the in vitro culture of pig epiblast cells. One such cell line, PICM-19, was isolated via colony-cloning and was found to spontaneously differentiate into hepati...

  19. Effects of Modeled Microgravity on Expression Profiles of Micro RNA in Human Lymphoblastoid Cells

    Science.gov (United States)

    Mangala, Lingegowda S.; Emami, Kamal; Story, Michael; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

    2010-01-01

    Among space radiation and other environmental factors, microgravity or an altered gravity is undoubtedly the most significant stress experienced by living organisms during flight. In comparison to the static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. Micro RNA (miRNA) has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. miRNA represents a class of single-stranded noncoding regulatory RNA molecules ( 22 nt) that control gene expressions by inhibiting the translation of mRNA to proteins. However, very little is known on the effect of altered gravity on miRNA expression. We hypothesized that the miRNA expression profile will be altered in zero gravity resulting in regulation of the gene expression and functional changes of the cells. To test this hypothesis, we cultured TK6 human lymphoblastoid cells in Synthecon s Rotary cell culture system (bioreactors) for 72 h either in the rotating (10 rpm) to model the microgravity in space or in the static condition. The cell viability was determined before and after culturing the cells in the bioreactor using both trypan blue and guava via count. Expressions of a panel of 352 human miRNA were analyzed using the miRNA PCRarray. Out of 352 miRNAs, expressions of 75 were significantly altered by a change of greater than 1.5 folds and seven miRNAs were altered by a fold change greater than 2 under the rotating culture condition. Among these seven, miR-545 and miR-517a were down regulated by 2 folds, whereas miR-150, miR-302a, miR-139-3p, miR-515-3p and miR-564 were up regulated by 2 to 8 folds. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA Illumina Microarray Analysis and validated the related genes using q-RT PCR.

  20. Dynamic microRNA profiles of hepatic differentiated human umbilical cord lining-derived mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Lina Cui

    Full Text Available Despite the extensive hepatic differentiation potential of human umbilical cord lining-derived mesenchymal stem cells (hUC-MSC, little is known about the molecular mechanisms of hUC-MSC differentiation. At the post-transcriptional level, microRNAs are key players in the control of cell fate determination during differentiation. In this study, we aimed to identify microRNAs involved in the hepatic differentiation of hUC-MSCs. After successfully isolating hUC- MSCs, we induced hepatocyte formation in vitro with growth factors. After 26 days of induction, hUC-MSCs could express hepatocyte-specific genes, synthesize urea and glycogen and uptake low-density lipoprotein. Cellular total RNA from hUC-MSCs and hepatic differentiated hUC-MSCs was collected at 7 time points, including 2 days, 6 days, 10 days, 14 days, 22 days and 26 days, for microRNA microarray analysis. Dynamic microRNA profiles were identified that did not overlap or only partially overlapped with microRNAs reported to be involved in human liver development, hepatocyte regeneration or hepatic differentiation of liver-derived progenitor cells. A total of 61 microRNAs among 1205 human and 144 human viral microRNAs displayed consistent changes and were altered at least 2-fold between hUC-MSCs and hepatic differentiated hUC-MSCs. Among these microRNAs, 25 were over-expressed; this over-expression occurred either gradually or increased sharply and was maintained at a high level. A total of 36 microRNAs were under-expressed, with an expression pattern similar to that of the over-expressed microRNAs. The expression of the altered expressed microRNAs was also confirmed by quantitative reverse-transcription polymerase chain reaction. We also found that microRNAs involved in hepatic differentiation were not enriched in hepatocyte or hepatocellular carcinoma cells and can potentially target liver-enriched transcription factors and genes. The elucidation of the microRNA profile during the hepatic

  1. Influence of vitamin D on cisplatin sensitivity in testicular germ cell cancer-derived cell lines and in a NTera2 xenograft model

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Blomberg Jensen, Martin; Nielsen, John Erik

    2013-01-01

    of pluripotency genes and simultaneous upregulation of the cell cycle regulators p21, p27, p53, p73 and FOXO1, while no significant effects were found in TCam-2 and 2102Ep cell lines (derived from seminoma and embryonal carcinoma, respectively). Anti-tumor effects of cholecalciferol, 1,25(OH)(2)D(3...

  2. Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture.

    Science.gov (United States)

    Vianney, John-Mary; Spitsbergen, John M

    2011-05-16

    Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent survival factor for both central and peripheral neurons. GDNF has been shown to be a potent survival factor for motor neurons during programmed cell death and continuous treatment with GDNF maintains hyperinnervation of skeletal muscle in adulthood. However, little is known about factors regulating normal production of endogenous GDNF in skeletal muscle. This study aimed to examine the role that motor neurons play in regulating GDNF secretion by skeletal muscle. A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma×neuroblastoma hybrid cells (NG108-15) were used to create nerve-muscle interactions in vitro. Acetylcholine receptors (AChRs) on nerve-myotube co-cultures were blocked with alpha-bungarotoxin (α-BTX). GDNF protein content in cells and in culture medium was analyzed by enzyme-linked immunosorbant assay (ELISA) and western blotting. GDNF localization was examined by immunocytochemistry. The nerve-muscle co-culture study indicated that the addition of motor neurons to skeletal muscle cells reduced the secretion of GDNF by skeletal muscle. The results also showed that blocking AChRs with α-BTX reversed the action of neural cells on GDNF secretion by skeletal muscle. Although ELISA results showed no GDNF in differentiated NG108-15 cells grown alone, immunocytochemical analysis showed that GDNF was localized in NG108-15 cells co-cultured with C2C12 myotubes. These results suggest that motor neurons may be regulating their own supply of GDNF secreted by skeletal muscle and that activation of AChRs may be involved in this process. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Acidosis-Induced Changes in Proteome Patterns of the Prostate Cancer-Derived Tumor Cell Line AT-1.

    Science.gov (United States)

    Ihling, Angelika; Ihling, Christian H; Sinz, Andrea; Gekle, Michael

    2015-09-04

    Under various pathological conditions, such as inflammation, ischemia and in solid tumors, physiological parameters (local oxygen tension or extracellular pH) show distinct tissue abnormalities (hypoxia and acidosis). For tumors, the prevailing microenvironment exerts a strong influence on the phenotype with respect to proliferation, invasion, and metastasis formation and therefore influences prognosis. In this study, we investigate the impact of extracellular metabolic acidosis (pH 7.4 versus 6.6) on the proteome patterns of a prostate cancer-derived tumor cell type (AT-1) using isobaric labeling and LC-MS/MS analysis. In total, 2710 proteins were identified and quantified across four biological replicates, of which seven were significantly affected with changes >50% and used for validation. Glucose transporter 1 and farnesyl pyrophosphatase were found to be down-regulated after 48 h of acidic treatment, and metallothionein 2A was reduced after 24 h and returned to control values after 48 h. After 24 and 48 h at pH 6.6, glutathione S transferase A3 and NAD(P)H dehydrogenase 1, cellular retinoic acid-binding protein 2, and Na-bicarbonate transporter 3 levels were found to be increased. The changes in protein levels were confirmed by transcriptome and functional analyses. In addition to the experimental in-depth investigation of proteins with changes >50%, functional profiling (statistical enrichment analysis) including proteins with changes >20% revealed that acidosis upregulates GSH metabolic processes, citric acid cycle, and respiratory electron transport. Metabolism of lipids and cholesterol biosynthesis were downregulated. Our data comprise the first comprehensive report on acidosis-induced changes in proteome patterns of a tumor cell line.

  4. Immunotherapy against Metastatic Melanoma with Human iPS Cell-Derived Myeloid Cell Lines Producing Type I Interferons.

    Science.gov (United States)

    Miyashita, Azusa; Fukushima, Satoshi; Nakahara, Satoshi; Kubo, Yosuke; Tokuzumi, Aki; Yamashita, Junji; Aoi, Jun; Haruta, Miwa; Senju, Satoru; Nishimura, Yasuharu; Jinnin, Masatoshi; Ihn, Hironobu

    2016-03-01

    In recent years, immunotherapy for advanced melanoma has been gaining increased attention. The efficacy of anti-cytotoxic T-lymphocyte antigen 4 antibodies, anti-programmed cell death 1 antibodies, and the BRAF(V600E) kinase inhibitor has been proven in metastatic melanoma. At the same time, adoptive cell transfer has significant effects against metastatic melanoma; however, it is difficult to apply on a broad scale because of the problems related to cell preparation. To overcome these problems, we developed immune cell therapy using induced pluripotent stem (iPS) cells. The benefit of our method is that a large number of cells can be readily obtained. We focused on macrophages for immune cell therapy because macrophage infiltration is frequently observed in solid cancers. In this study, the efficacy of human iPS cell-derived myeloid cell lines (iPS-ML) genetically modified to express type I IFNs against human melanoma cells was examined. The morphology, phagocytic ability, and surface markers of iPS-ML were similar to those of macrophages. The iPS-ML that express type I IFNs (iPS-ML-IFN) showed significant effects in inhibiting the growth of disseminated human melanoma cells in SCID mice. The infiltration of iPS-ML into the tumor nests was confirmed immunohistologically. The iPS-ML-IFNs increased the expression of CD169, a marker of M1 macrophages that can activate antitumor immunity. The iPS-ML-IFNs could infiltrate into tumor tissue and exert anticancer effects in the local tumor tissue. In conclusion, this method will provide a new therapeutic modality for metastatic melanoma.

  5. INCREASED EXPRESSION OF GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR IN RAT BRAIN AFTER TRAUMATIC BRAIN INJURY

    Directory of Open Access Journals (Sweden)

    V. Rahimi-Movaghar

    2005-04-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF plays important roles not only for the differentiation of neurons during normal development but also for the survival and recovery of many populations of mature neurons. The effect of traumatic brain injury (TBI on the expression of GDNF is currently unknown. To determine if there is alteration in GDNF after TBI we examined the effect of controlled cortical impact (CCI injury on GDNF protein levels at 6 hours, 1 day, 1 week, and 4 weeks following injury by utilizing a commercially available antibody specific to GDNF. Rats were anesthetized and surgically prepared for CCI injury (4 m/sec, 2.7 mm and sham surgery. Injured and sham animals (n=6 per group were sacrificed at 6 hours, 1 day, 1 week, and 4 weeks and perfused with 4% paraformaldehyde. Coronal sections (35 mm thick were cut through the hippocampus. An increased expression of GDNF protein was observed by immunohistochemistry in the dentate gyrus of hippocampus and the cortex in injured rats compared to sham controls. The increased expression of GDNF was more evidently observed in the ipsilateral dentate gyrus and the area around the contusion in the cortex. In the cortex, GDNF immunoreactivity appeared greatest in cells with glial morphology but in the hippocampus, GDNF immunoreactivity was greatest in neuronal-like cells. These changes were observed at 1 day, 1 and 4 weeks postinjury. We speculate that the up-regulation of the GDNF protein may reflect its neurotrophic and neuroprotective effect on dopaminergic system responding to the TBI insult.

  6. MicroRNA regulation of central glial cell line-derived neurotrophic factor (GDNF) signalling in depression.

    Science.gov (United States)

    Maheu, M; Lopez, J P; Crapper, L; Davoli, M A; Turecki, G; Mechawar, N

    2015-02-17

    Although multiple studies have reported that peripheral glial cell line-derived neurotrophic factor (GDNF) is reduced in depression, cerebral GDNF signalling has yet to be examined in this condition. Here, we report an isoform-specific decrease in GDNF family receptor alpha 1 (GFRA1) mRNA expression, resulting in lowered GFRα1a protein levels in basolateral amygdala (BLA) samples from depressed subjects. Downregulation of GFRα1a was associated with increased expression of microRNAs, including miR-511, predicted to bind to long 3' untranslated region (3'-UTR)-containing transcripts (GFRA1-L) coding for GFRα1a. Transfection of human neural progenitor cells (NPCs) with a miR-511 mimic was sufficient to repress GFRA1-L/GFRα1a without altering GFRα1b, and resulted in pathway-specific changes in immediate early gene activity. Unexpectedly, GFRα1a knockdown did not reduce NPC responses to GDNF. Rather, it greatly enhanced mitogen-activated protein kinase signalling. This effect appeared to be mediated by GDNF/soluble GFRα1/neural cell adhesion molecule binding, and substituting the soluble GFRα1a/GFRα1b content of miR-511-transfected NPCs with that of controls rescued signalling. In light of previous reports suggesting that GFRα1b can inhibit GFRα1a-induced neuroplasticity, we also assessed the association between GFRα1 and doublecortin (DCX; a hyperplastic marker) in human BLA. Although controls displayed coordinated expression of GFRα1a and b isoforms and these correlated positively with DCX, the only significant association observed among depressed subjects was a strongly negative correlation between GFRα1b and DCX. Taken together, these results suggest that microRNA-mediated reductions of GFRα1a in depression change the quality, rather than the quantity, of GDNF signalling. They also suggest that central GDNF signalling may represent a novel target for antidepressant treatment.

  7. Imipramine activates glial cell line-derived neurotrophic factor via early growth response gene 1 in astrocytes.

    Science.gov (United States)

    Kim, Yeni; Kim, Se Hyun; Kim, Yong Sik; Lee, Young Han; Ha, Kyooseob; Shin, Soon Young

    2011-06-01

    Recent evidence has suggested that deficits in glial plasticity contribute to the pathophysiology of depressive disorders. The present study explored early growth response 1 (EGR-1) transcriptional regulation of imipramine-induced glial cell line-derived neurotrophic factor (GDNF) expression in astrocytes. After we observed the induction of GDNF mRNA expression in rat astrocytes in response to imipramine, deletion mutant studies showed that the proximal region between -493 and -114 of the GDNF promoter, which contains three binding sites for EGR-1, was essential for maximal imipramine-induced activation of GDNF promoter. The dose-dependent upregulation of EGR-1 by imipramine, the activation of GDNF by the over-expression of EGR-1 without imipramine and the reduction in the imipramine-induced GDNF mRNA expression after silencing of endogenous EGR-1 demonstrated that EGR-1 is upregulated by imipramine to activate the GDNF promoter. Furthermore, imipramine-induced GDNF mRNA expression was strongly attenuated in primary astrocytes from Egr-1(-/-) mice, and the immunoreactivity to an anti-GDNF antibody in glial fibrillary acidic protein-positive cells was lower in imipramine-treated astrocytes from Egr-1(-/-) mice than in those from Egr-1(+/-) mice. To determine whether mitogen-activated protein kinases (MAPKs) were associated with imipramine-induced EGR-1 expression, we examined the induction of MAPK phosphorylation in response to imipramine. Pretreatment of rat primary astrocytes with the MAPK kinase inhibitor U0126 or the JNK inhibitor SP600125 strongly inhibited imipramine-stimulated EGR-1 expression. In conclusion, we found that imipramine induction of EGR-1 upregulated GDNF in astrocytes in a dose-dependent manner. This upregulation may occur through the MEK/ERK and JNK MAPK pathways, which suggests a new therapeutic mechanism of action for depressive disorders.

  8. Distribution of glial cell line-derived neurotrophic factor receptor alpha-1 in the brain of adult zebrafish.

    Science.gov (United States)

    Lucini, Carla; Carla, Lucini; Facello, Bruna; Bruna, Facello; Maruccio, Lucianna; Lucianna, Maruccio; Langellotto, Fernanda; Fernanda, Langellotto; Sordino, Paolo; Paolo, Sordino; Castaldo, Luciana; Luciana, Castaldo

    2010-08-01

    Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor for several types of neurons in the central and peripheral nervous systems. The biological activity of GDNF is mediated by a multicomponent receptor complex that includes a common transmembrane signaling component (the rearranged during transfection (RET) proto-oncogene product, a tyrosine kinase receptor) as well as a GDNF family receptor alpha (GFRalpha) subunit, a high-affinity glycosyl phosphatidylinositol (GPI)-linked binding element. Among the four known GFRalpha subunits, GFRalpha1 preferentially binds to GDNF. In zebrafish (Danio rerio) embryos, the expression of the GFRalpha1a and GFRalpha1b genes has been shown in primary motor neurons, the kidney, and the enteric nervous system. To examine the activity of GFRalpha in the adult brain of a lower vertebrate, we have investigated the localization of GFRalpha1a and GFRalpha1b mRNA and the GFRalpha1 protein in zebrafish. GFRalpha1a and GFRalpha1b transcripts were observed in brain extracts by reverse transcription-polymerase chain reaction. Whole-mount in-situ hybridization experiments revealed a wide distribution of GFRalpha1a and GFRalpha1b mRNAs in various regions of the adult zebrafish brain. These included the olfactory bulbs, dorsal and ventral telencephalic area (telencephalon), preoptic area, dorsal and ventral thalamus, posterior tuberculum and hypothalamus (diencephalon), optic tectum (mesencephalon), cerebellum, and medulla oblongata (rhombencephalon). Finally, expression patterns of the GFRalpha1 protein, detected immunohistochemically, correlated well with the mRNA expression and provided further insights into translational activity at the neuroanatomical level. In conclusion, the current study demonstrated that the presence of GFRalpha1 persists beyond the embryonic development of the zebrafish brain and, together with the GDNF ligand, is probably implicated in the brain physiology of an adult teleost fish.

  9. Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Margarita Chumarina

    2017-03-01

    Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

  10. Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays

    Energy Technology Data Exchange (ETDEWEB)

    Vares, Guillaume, E-mail: vares@nirs.go.jp [National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555 (Japan); Wang, Bing, E-mail: jp2813km@nirs.go.jp [National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555 (Japan); Tanaka, Kaoru; Kakimoto, Ayana; Eguchi-Kasai, Kyomi; Nenoi, Mitsuru [National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555 (Japan)

    2011-01-10

    The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1 Gy X-rays, followed 6 h later by challenging 1 Gy heavy-ion radiation (carbon-ion: 20 and 40 keV/{mu}m, neon-ion: 150 keV/{mu}m). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.

  11. Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays.

    Science.gov (United States)

    Varès, Guillaume; Wang, Bing; Tanaka, Kaoru; Kakimoto, Ayana; Eguchi-Kasai, Kyomi; Nenoi, Mitsuru

    2011-01-10

    The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.

  12. Effects of inorganic and organic arsenic compounds on growth and apoptosis of human T-lymphoblastoid leukemia cells.

    Science.gov (United States)

    Hikita, Eri; Arai, Mariko; Tanaka, Sachiko; Onda, Kenji; Utsumi, Hiroya; Yuan, Bo; Toyoda, Hiroo; Hirano, Toshihiko

    2011-12-01

    To investigate the effects of inorganic and organic arsenic compounds on human T-lymphoblastoid leukemia cells. Cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5¬diphenyltetrazolium bromide (MTT) assay. Apoptotic cell morphology was examined by cell staining with Hoechst 33342. Cellular caspase-3/7 activities were measured after arsenic treatment. The inhibitory concentration by 50% (IC(50)) values of As(2)O(3) towards MOLT-4 and daunorubicin- resistant MOLT-4/DNR cell proliferation were 0.87 and 0.92 μM, while the values for arsenic acid were 69.1 and 116.6 μM, respectively. These arsenic compounds also inhibited mitogen-induced proliferation of human peripheral blood mononuclear cells. Six organic arsenic compounds did not inhibit leukemia cell proliferation. As(2)O(3) and arsenic acid induced apoptotic cell morphology and increased caspase-3/7 activity in the leukemia cells. Ascorbic acid and buthionine sulfoxide enhanced, while N-acetyl-L-cysteine abated, the suppressive effects of inorganic arsenic compounds on leukemia cell proliferation. As(2)O(3) and arsenic acid inhibit proliferation and induce apoptosis in MOLT-4 and daunorubicine-resistant MOLT-4/DNR cells via glutathione-depletion and subsequent caspase-3/7 activation. Organic arsenic compounds have no inhibitory activity on the leukemia cell proliferation. Inorganic arsenic compounds are suggested as useful agents for treatment of T-lymphoblastoid leukemia.

  13. Comparative study on the cytotoxic effects of benzalkonium chloride on the Wong-Kilbourne derivative of Chang conjunctival and IOBA-NHC cell lines

    Science.gov (United States)

    Brasnu, E.; Brignole-Baudouin, F.; Riancho, L.; Warnet, J.-M.

    2008-01-01

    Purpose The Wong-Kilbourne derivative of Chang conjunctiva-derived cell line has been widely used for toxicological and functional in vitro studies on the ocular surface. The common reserve to this cell line is the reported contamination with HeLa cells. Thus, the IOBA-NHC spontaneously immortalized conjunctival epithelial cell line has been recently developed and did not show other cell type contamination. Our purpose was to determine whether both cell lines would be equally suitable for in vitro toxicological studies. Therefore, we compared in these two cell types the toxic effects of the preservative, benzalkonium chloride (BAC); its toxicity has been often reported on conjunctival in vivo and in vitro models. Methods The necrotic, apoptotic, and oxidative effects of BAC were evaluated on Chang and IOBA-NHC cell lines using microplate cytofluorometry tests (neutral red, 2,7- dichlorofluorescein diacetate dye [H2DCF-DA], hydroethidine, and Yopro-1), flow cytometry (Annexin V/7-AAD and DNA content tests), and standard immunofluorescence stainings. Cells were exposed to five concentrations of BAC (10−2%, 5.10−3%, 10−3%, 10−4%, and 10−5%) for two incubation times: 15 min of treatment and 15 min of treatment followed by 24 h of cell recovery in complete medium. Results All parameters of toxicity increased in a BAC dose-dependent manner on both cell lines. Conclusions The comparison of BAC toxicity on both cell lines supported the use of IOBA-NHC and Chang cells for toxicological in vitro studies. Drawbacks of both cell lines have to be known and considered in studies performed on these cell lines. PMID:18334956

  14. Morphological Structure and Transcriptome Comparison of the Cytoplasmic Male Sterility Line in Brassica napus (SaNa-1A) Derived from Somatic Hybridization and Its Maintainer Line SaNa-1B

    Science.gov (United States)

    Du, Kun; Liu, Qier; Wu, Xinyue; Jiang, Jinjin; Wu, Jian; Fang, Yujie; Li, Aimin; Wang, Youping

    2016-01-01

    SaNa-1A is a novel cytoplasmic male sterility (CMS) line in Brassica napus derived from progenies of somatic hybrids between B.napus and Sinapis alba, and SaNa-1B is the corresponding maintainer line. In this study, phenotypic differences of floral organs between CMS and the maintainer lines were observed. By microscope observation in different anther developmental stages of two lines, we found the anther development in SaNa-1A was abnormal since the tetrad stage, and microspore development was ceased during the uninucleate stage. Transcriptomic sequencing for floral buds of sterile and fertile plants were conducted to elucidate gene expression and regulation caused by the alien chromosome and cytoplasm from S. alba. Clean tags obtained were assembled into 195,568 unigenes, and 7811 unigenes distributed in the metabolic and protein synthesis pathways were identified with significant expression differences between two libraries. We also observed that genes participating in carbon metabolism, tricarboxylic acid cycle, oxidative phosphorylation, oxidation–reduction system, pentatricopeptide repeat, and anther development were downregulated in the sterile line. Some of them are candidates for researches on the sterility mechanism in the CMS material, fertility restoration, and improvement of economic traits in the maintainer line. Further research on the tags with expressional specificity in the fertile line would be helpful to explore desirable agronomic traits from wild species of rapeseed. PMID:27656189

  15. The prothrombin time/international normalised ratio (PT/INR) line: derivation of local INR with commercial thromboplastins and coagulometers – two independent studies

    DEFF Research Database (Denmark)

    Poller, Leon; Ibrahim, S.; Keown, M.

    2011-01-01

    Background: The WHO scheme for prothrombin time (PT) standardization has been limited in application, because of its difficulties in implementation, particularly the need for mandatory manual PT testing and for local provision of thromboplastin international reference preparations (IRP). Methods...... thromboplastins and coagulometers. INRs were compared with manual certified values with thromboplastin IRP from expert centres and in the second study also with INRs from local ISI calibrations. Results: In the first study with the PT/INR Line, 8.7% deviation from certified INRs was reduced to 1.1% with human.......5% with bovine/combined reagents with local ISI calibrations and to 2.9% with the PT/INR Line. Mean INR dispersion was reduced with all thromboplastins and automated systems using the PT/INR Line. Conclusions: The procedure using the PT/INR Line provides reliable INR derivation without the need for WHO ISI...

  16. Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial, yeast and human lymphoblastoid cells.

    Science.gov (United States)

    Andrews, J; Abd-Ellah, M F; Randolph, N L; Kenworthy, K E; Carlile, D J; Friedberg, T; Houston, J B

    2002-11-01

    1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters K(m) or S(50), V(max) and intrinsic clearance (CL(int)) or maximum clearance (CL(max)) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The K(m) or S(50) for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the V(max) for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CL(int) or CL(max). In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.

  17. Mutagenicity and clastogenicity of extracts of Helicobacter pylori detected by the Ames test and in the micronucleus test using human lymphoblastoid cells.

    Science.gov (United States)

    Arimoto-Kobayashi, Sakae; Ohta, Kaori; Yuhara, Yuta; Ayabe, Yuka; Negishi, Tomoe; Okamoto, Keinosuke; Nakajima, Yoshihiro; Ishikawa, Takeshi; Oguma, Keiji; Otsuka, Takanao

    2015-07-01

    Epidemiological studies have demonstrated a close association between infection with Helicobacter pylori (H.pylori) and the development of gastric carcinoma. Chronic H.pylori infection increases the frequency of mutation in gastric epithelial cells. However, the mechanism by which infection of H.pylori leads to mutation in gastric epithelial cells is unclear. We suspected that components in H.pylori may be related to the mutagenic response associated with DNA alkylation, and could be detected with the Ames test using a more sensitive strain for alkylating agents. Our investigation revealed that an extract of H.pylori was mutagenic in the Ames test with Salmonella typhimurium YG7108, which is deficient in the DNA repair of O(6)-methylguanine. The extract of H.pylori may contain methylating or alkylating agents, which might induce O (6)-alkylguanine in DNA. Mutagenicity of the alkylating agents N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N-nitrosoguanidine in the Ames test with S.typhimurium TA1535 was enhanced significantly in the presence of the extract of H.pylori. The tested extracts of H.pylori resulted in a significant induction of micronuclei in human-derived lymphoblastoid cells. Heat instability and dialysis resistance of the extracts of H.pylori suggest that the mutagenic component in the extracts of H.pylori is a heat-unstable large molecule or a heat-labile small molecule strongly attached or adsorbed to a large molecule. Proteins in the extracts of H.pylori were subsequently fractionated using ammonium sulphate precipitation. However, all fractions expressed enhancing effects toward MNU mutagenicity. These results suggest the mutagenic component is a small molecule that is absorbed into proteins in the extract of H.pylori, which resist dialysis. Continuous and chronic exposure of gastric epithelial cells to the alkylative mutagenic component from H.pylori chronically infected in the stomach might be a causal factor in the gastric carcinogenesis

  18. Deriving the Extinction to Young Stellar Objects using [Fe II] Near-infrared Emission Lines: Prescriptions from GIANO High-resolution Spectra

    Science.gov (United States)

    Pecchioli, T.; Sanna, N.; Massi, F.; Oliva, E.

    2016-07-01

    The near-infrared (NIR) emission lines of Fe+ at 1.257, 1.321, and 1.644 μm share the same upper level; their ratios can then be exploited to derive the extinction to a line emitting region once the relevant spontaneous emission coefficients are known. This is commonly done, normally from low-resolution spectra, in observations of shocked gas from jets driven by Young Stellar Objects. In this paper we review this method, provide the relevant equations, and test it by analyzing high-resolution (R ∼ 50,000) NIR spectra of two young stars, namely the Herbig Be star HD 200775 and the Be star V1478 Cyg, which exhibit intense emission lines. The spectra were obtained with the new GIANO echelle spectrograph at the Telescopio Nazionale Galileo. Notably, the high-resolution spectra allowed checking the effects of overlapping telluric absorption lines. A set of various determinations of the Einstein coefficients are compared to show how much the available computations affect extinction derivation. The most recently obtained values are probably good enough to allow reddening determination within 1 visual mag of accuracy. Furthermore, we show that [Fe ii] line ratios from low-resolution pure emission-line spectra in general are likely to be in error due to the impossibility to properly account for telluric absorption lines. If low-resolution spectra are used for reddening determinations, we advice that the ratio 1.644/1.257, rather than 1.644/1.321, should be used, being less affected by the effects of telluric absorption lines.

  19. Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations.

    Science.gov (United States)

    Momcilovic, Olga; Sivapatham, Renuka; Oron, Tal Ronnen; Meyer, Morten; Mooney, Sean; Rao, Mahendra S; Zeng, Xianmin

    2016-01-01

    We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight of these lines (one of each SNCA, LRRK2 and GBA, four PARK2 lines, and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line, nor amongst the patient lines, although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines. We performed whole genome expression analyses of the lines at each stage of differentiation (fibroblast, iPSC, NSC, and dopaminergic culture) in an attempt to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation, no mutation-specific clustering or difference was observed, though consistent changes in patient lines were detected in genes associated mitochondrial biology. We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line, and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may not be sufficient to determine the cause or mechanism of the disease, and highlights the need to use more focused strategies for large-scale data analysis.

  20. Synthesis and cytotoxic effect on cancer cell lines and macrophages of novel progesterone derivatives having an ester or a carbamate function at C-3 and C-17.

    Science.gov (United States)

    Chávez-Riveros, Alejandra; Garrido, Mariana; Ramírez Apan, María Teresa; Zambrano, Armando; Díaz, Mario; Bratoeff, Eugene

    2014-07-23

    In this study we report the cytotoxic effect on human cancer cells of two series of novel progesterone derivatives; the first containing an aromatic ester (8a-e) or a carbamate functions both linked to C-3 (9a-e) on the pregn-4,16-diene-6,20-dione skeleton. In the second series, both functional groups (ester and carbamate) are bound to C-17 on the pregn-4,6-diene-3,20-dione scaffold (13a-e and 14a-e). The panel cancer cell lines used in this study were the following: PC-3 (human prostate cancer cell line), MCF-7 (human breast cancer cell line), HCT-15 (human colon cancer cell line) and J774 (noncancerous murine macrophages) for comparison. The results from this study showed that steroid 14a, having a carbamate function at C-17, was the most potent against PC-3 cell line (96.6%) while 8c and 8e showed much higher cytotoxic activity (100%) for MCF-7 cell line. Finally, compounds 8c and 14a displayed selective properties towards tumor cell lines than noncancerous murine macrophages. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Preservation of biological activity of glial cell line-derived neurotrophic factor (GDNF) after microencapsulation and sterilization by gamma irradiation.

    Science.gov (United States)

    Checa-Casalengua, P; Jiang, C; Bravo-Osuna, I; Tucker, B A; Molina-Martínez, I T; Young, M J; Herrero-Vanrell, R

    2012-10-15

    A main issue in controlled delivery of biotechnological products from injectable biodegradable microspheres is to preserve their integrity and functional activity after the microencapsulation process and final sterilization. The present experimental work tested different technological approaches to maintain the biological activity of an encapsulated biotechnological product within PLGA [poly (lactic-co-glycolic acid)] microspheres (MS) after their sterilization by gamma irradiation. GDNF (glial cell line-derived neurotrophic factor), useful in the treatment of several neurodegenerative diseases, was chosen as a labile model protein. In the particular case of optic nerve degeneration, GDNF has been demonstrated to improve the damaged retinal ganglion cells (RGC) survival. GDNF was encapsulated in its molecular state by the water-in-oil-in-water (W/O/W) technique or as solid according to the solid-in-oil-in-water (S/O/W) method. Based on the S/O/W technique, GDNF was included in the PLGA microspheres alone (S/O/W 1) or in combination with an antioxidant (vitamin E, Vit E) (S/O/W 2). Microspheres were sterilized by gamma-irradiation (dose of 25 kGy) at room and low (-78 °C) temperatures. Functional activity of GDNF released from the different microspheres was evaluated both before and after sterilization in their potential target cells (retinal cells). Although none of the systems proposed achieved with the goal of totally retain the structural stability of the GDNF-dimer, the protein released from the S/O/W 2 microspheres was clearly the most biologically active, showing significantly less retinal cell death than that released from either W/O/W or S/O/W 1 particles, even in low amounts of the neurotrophic factor. According to the results presented in this work, the biological activity of biotechnological products after microencapsulation and sterilization can be further preserved by the inclusion of the active molecule in its solid state in combination with

  2. Comparative Mapping of the Oregon Wolfe Barley Using Doubled Haploid Lines Derived from Female and Male Gametes

    Science.gov (United States)

    The Oregon Wolfe Barley mapping population is a resource for genetics research and instruction. Prior reports are based on a population of doubled haploid (DH) lines developed by the Hordeum bulbosum (H.b.) method, which samples female gametes. We developed new DH lines from the same cross using ant...

  3. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

  4. Genomic in situ hybridization analysis of Thinopyrum chromatin in a wheat-Th. intermedium partial amphiploid and six derived chromosome addition lines

    Science.gov (United States)

    Chen; Conner; Laroche; Ji; Armstrong; Fedak

    1999-12-01

    The genomic origin of alien chromosomes present in a wheat-Thinopyrum intermedium partial amphiploid TAF46 (2n = 8x = 56) and six derived chromosome addition lines were analyzed by genomic in situ hybridization (GISH) using S genomic DNA from Pseudoroegneria strigosa (2n = 2x = 14, SS) as a probe. The GISH analysis clearly showed that the chromosome complement of the partial amphiploid TAF46 consists of an entire wheat genome plus one synthetic genome consisting of a mixture of six S genome chromosomes and eight J (=E) genome chromosomes derived from Th. intermedium (2n = 6x = 42, JJJ(s)J(s)SS). There were no Js genome chromosomes present in TAF46. The J genome chromosomes present in TAF46 displayed a unique GISH hybridization pattern with the S genomic DNA probe, in which S genome DNA strongly hybridized at the terminal regions and weakly hybridized over the remaining parts of the chromosomes. This provides a diagnostic marker for distinguishing J genome chromosomes from Js or S genome or wheat ABD genome chromosomes. The genomic origin of the alien chromosomes present in the six derived chromosome addition lines were identified by their characteristic GISH hybridization patterns with S genomic DNA probe. GISH analysis showed that addition lines L1, L2, L3, and L5 carried one pair of J genome chromosomes, while addition lines L4 and L7 each carried one pair of S genome chromosomes. GISH patterns detected by the S genome probe on addition line of L1 were identical to those of the J genome chromosomes present in the partial amphiploid TAF46, suggesting that these chromosomes were not structurally altered when they were transferred from TAF46 to addition lines.

  5. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  6. Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD for monogenic disorder

    Directory of Open Access Journals (Sweden)

    Abdelkrim Hmadcha

    2016-05-01

    Full Text Available From 106 human blastocyts donate for research after in vitro fertilization (IVF and preimplantation genetic diagnosis (PGD for monogenetic disorder, 3 human embryonic stem cells (hESCs HVR1, HVR2 and HVR3 were successfully derived. HVR1 was assumed to be genetically normal, HVR2 carrying Becker muscular dystrophy and HVR3 Hemophilia B. Despite the translocation t(9;15(q34.3;q14 detected in HVR2, all the 3 cell lines were characterised in vitro and in vivo as normal hESCs lines and were registered in the Spanish Stem Cell Bank.

  7. Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

    Institute of Scientific and Technical Information of China (English)

    SHI Cheng; SHEN Huan; JIANG Wei; SONG Zhi-hua; WANG Cheng-yan; WEI Li-hui

    2011-01-01

    Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background,which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use,especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.Methods Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propogate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.Results We generated a new Chinese human embryonic stem cells line,CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines:normal morphology,karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.Conclusions This newly established Chinese cell line,CH1,which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells,will provide

  8. Cytokines and Epstein Barr virus (EBV) genes expression in blood chronic lymphocytic leukaemia (CLL) cells and their immortalised CLL cell lines.

    Science.gov (United States)

    Laytragoon-Lewin, Nongnit; Chen, Fu; Castro, Juan; Avila-Carino, Javier; Lewin, Freddi

    2003-01-01

    We have encountered two unique chronic lymphocytic leukaemia (CLL) patients, PG and NN. Some blood CLL cells of these patients have been infected and carry Epstein Barr virus (EBV) in vivo. In spite of their early-activated G0/G1 stage of post germinal center (GC) memory cells, ex vivo EBV-carrying blood CLL cells of PG clone expressed LMPs and used specific QUK splice for their EBNA1 expression, similar to the EBV-carrying cells of non-B origin. Interestingly, EBV-carrying CLL cells of NN clone expressed LMP2a and used UK-splice for their EBNA1 expression, similar to the in vivo EBV-carrying high density normal B cells in the blood of healthy individuals. The CLL-derived lines but not normal lymphoblastoid cell line (LCL) used QUK- and YUK-splice for their EBNA1 expression. As expected, LCL and their permanent CLL-derived lines used Cp promoter and up-regulated their EBNA2 expression. Blood CLL cells and the CLL-derived cell lines of these patients spontaneously produced cytokines as shown by microarray assay. The types and quantities of cytokines might relate to their CLL origin and viral strain in the given CLL cells. Neither blood CLL nor their CLL-derived cell lines express any detectable apoptosis-inducer ligands, CD95L or Apo 3L. As a consequence of cell cycle progression, CLL-derived cell lines up-regulated their co-stimulator molecules CD80 and apoptosis-related receptor CD95. Since only the rare EBV-carrying CLL cells grew in vitro, the combination of viral genome and cytokines seems to be critical for the outgrowth of EBV-carrying CLL cells over their EBV-negative counterpart in vitro but not in vivo.

  9. A duplicated coxI gene is associated with cytoplasmic male sterility in an alloplasmic Brassica juncea line derived from somatic hybridization with Diplotaxis catholica

    Indian Academy of Sciences (India)

    Aruna Pathania; Rajesh Kumar; V. Dinesh Kumar; Ashutosh; K. K. Dwivedi; P. B. Kirti; P. Prakash; V. L. Chopra; S. R. Bhat

    2007-08-01

    A cytoplasmic male sterile (CMS) line of Brassica juncea was derived by repeated backcrossing of the somatic hybrid (Diplotaxis catholica + B. juncea) to B. juncea. The new CMS line is comparable to euplasmic lines for almost all characters, except for flowers which bear slender, needle-like anthers with aborted pollen. Detailed Southern analysis revealed two copies of coxI gene in the CMS line. One copy, coxI-1 is similar to the coxI gene of B. juncea, whereas the second copy, coxI-2 is present in a novel rearranged region. Northern analysis with eight mitochondrial gene probes showed altered transcript pattern only for the coxI gene. Two transcripts of 2.0 and 2.4 kb, respectively, were detected in the CMS line. The novel 2.4 kb transcript was present in floral bud tissue but absent in the leaf tissue. In plants where male sterility broke down under high temperature during the later part of the growing season, the 2.4 kb coxI transcript was absent, which suggested its association with the CMS. The two coxI genes from the CMS line showed two amino acid changes in the coding region. The novel coxI gene showed unique repeats in the 5′ region suggesting recombination of mitochondrial genomes of the two species. The possible role of the duplicated coxI gene in causing male sterility is discussed.

  10. Relationships among the slopes of lines derived from various data analysis techniques and the associated correlation coefficient

    Science.gov (United States)

    Cohen, S. C.

    1980-01-01

    A technique for fitting a straight line to a collection of data points is given. The relationships between the slopes and correlation coefficients, and between the corresponding standard deviations and correlation coefficient are given.

  11. Activation of vitamin D receptor (VDR)- and peroxisome proliferator-activated receptor (PPAR)-signaling pathways through 1,25(OH)(2)D(3) in melanoma cell lines and other skin-derived cell lines.

    Science.gov (United States)

    Sertznig, Pit; Seifert, Markus; Tilgen, Wolfgang; Reichrath, Jörg

    2009-07-01

    We have investigated expression of vitamin D receptor (VDR) and peroxisome proliferator-activated receptors (PPAR)alpha, delta, gamma in primary cultured normal melanocytes (NHM), melanoma cell lines (MeWo, SK-Mel-5, SK-Mel-25, SK-Mel-28), a cutaneous squamous cell carcinoma cell line (SCL-1) and an immortalized sebocyte cell line (SZ95). LNCaP prostate cancer cells, MCF-7 breast cancer cells and embryonic kidney cells (HEK-293) were used as controls. VDR and PPAR mRNA were detected, quantitated and compared in these cell lines using real-time quantitative polymerase chain reaction (RTqPCR). The expression patterns of these nuclear receptors (NRs) varied strongly between the different cell lines according to their origin. PPARdelta and PPARgamma were less strongly expressed in the melanoma cell lines and in the other skin-derived cell lines as compared to the control cell lines. PPARalpha and VDR were stronger expressed in the 1,25(OH)(2)D(3)-sensitive melanoma cells (MeWo and in SK-Mel-28) than in the 1,25(OH)(2)D(3)-resistent melanoma cell lines (SK-Mel-5 and SK-Mel-25) or in NHM. Interestingly, VDR expression was increased by the treatment with 1,25(OH)(2)D(3) in 1,25(OH)(2)D(3)-sensitive melanoma cells but not in 1,25(OH)(2)D(3)-resistent melanoma cell lines. 1,25(OH)(2)D(3) increased the expression of PPARalpha in almost all cell lines analyzed. Our results indicate a cross-talk between VDR- and PPAR-signaling pathways in various cell types including melanoma cells. Further investigations are required to investigate the physiological and pathophysiological relevance of this cross-talk. Because VDRand PPAR-signaling pathways regulate a multitude of genes that are of importance for a multitude of cellular functions including cell proliferation, cell differentiation, immune responses and apoptosis, the provided link between VDR and PPAR may open important new perspectives for treatment and prevention of melanoma and other diseases.

  12. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

    Science.gov (United States)

    Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

  13. The gene coding for glial cell line derived neurotrophic factor (GDNF) maps to chromosome 5p12-p13.1

    Energy Technology Data Exchange (ETDEWEB)

    Schindelhauer, D.; Schuffenhauer, S.; Meitinger, T. [Maximiland-Universitaet, Munich (Germany)] [and others

    1995-08-10

    The gene coding for glial cell line derived neurotrophic factor (GDNF) has biological properties that may have potential as a treatment for Parkinson`s and motoneuron diseases. Using the NIGMS Mapping Panel 2, we have localized the GDNF gene to human chromosome 5p12-p13.1. Large NruI and NotI fragments on chromosome 5 will facilitate the construction of a long-range map of the region. 26 refs., 1 fig., 1 tab.

  14. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

    OpenAIRE

    Katt, Moriah E.; Xu, Zinnia S.; Gerecht, Sharon; Searson, Peter C.

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial...

  15. Synthesis and Pharmacophore Modelling of 2,6,9-Trisubstituted Purine Derivatives and Their Potential Role as Apoptosis-Inducing Agents in Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Jeannette Calderón-Arancibia

    2015-04-01

    Full Text Available A series of 2,6,9-trisubstituted purine derivatives have been synthesized and investigated for their potential role as antitumor agents. Twelve compounds were obtained by a three step synthetic procedure using microwave irradiation in a pivotal step. All compounds were evaluated in vitro to determine their potential effect on cell toxicity by the MTT method and flow cytometry analysis on four cancer cells lines and Vero cells. Three out of twelve compounds were found to be promising agents compared to a known and effective anticancer drug, etoposide, in three out of four cancer cell lines assayed with considerable selectivity. Preliminary flow cytometry data suggests that compounds mentioned above induce apoptosis on these cells. The main structural requirements for their activity for each cancer cell line were characterized with a preliminary pharmacophore model, which identified aromatic centers, hydrogen acceptor/donor center and a hydrophobic area. These features were consistent with the cytotoxic activity of the assayed compounds.

  16. A transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins through N-glycosylation.

    Science.gov (United States)

    Hu, Jia-Biao; Zhang, Peng; Wang, Mei-Xian; Zhou, Fang; Niu, Yan-Shan; Miao, Yun-Gen

    2012-08-01

    Glycoproteins have been implicated in a wide variety of important biochemical and biological functions, including protein stability, immune function, enzymatic function, cellular adhesion and others. Unfortunately, there is no therapeutic protein produced in insect system to date, due to the expressed glycoproteins are paucimannosidic N-glycans, rather than the complex, terminally sialylated N-glycans in mammalian cells. In this paper, we cloned the necessary genes in glycosylation of mammalian cells, such as N-acetylglucosaminyltransferase II (Gn-TII), galactosyltransferases (Gal-Ts), 2,6-Sial-T (ST6 GalII)and 2,3-Sial-T (ST3GalIII), and transformed them to silkworm genome of BmN cell line through transgenesis to establish a transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins. The study supplied a new insect cell line which is practically to produce "bisected" complex N-glycans like in mammalian cells.

  17. Role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas.

    Directory of Open Access Journals (Sweden)

    Maurizio Sorice

    Full Text Available We previously found that a directional movement of the raft component GD3 towards mitochondria, by its association with microtubules, was mandatory to late apoptogenic events triggered by CD95/Fas. Since CLIPR-59, CLIP-170-related protein, has recently been identified as a microtubule binding protein associated with lipid rafts, we analyzed the role of GD3-CLIPR-59 association in lymphoblastoid T cell apoptosis triggered by CD95/Fas. To test whether CLIPR-59 could play a role at the raft-microtubule junction, we performed a series of experiments by using immunoelectron microscopy, static or flow cytometry and biochemical analyses. We first assessed the presence of CLIPR-59 molecule in lymphoblastoid T cells (CEM. Then, we demonstrated that GD3-microtubule interaction occurs via CLIPR-59 and takes place at early time points after CD95/Fas ligation, preceding the association GD3-tubulin. GD3-CLIPR-59 association was demonstrated by fluorescence resonance energy transfer (FRET analysis. The key role of CLIPR-59 in this dynamic process was clarified by the observation that silencing CLIPR-59 by siRNA affected the kinetics of GD3-tubulin association, spreading of GD3 towards mitochondria and apoptosis execution. We find that CLIPR-59 may act as a typical chaperone, allowing a prompt interaction between tubulin and the raft component GD3 during cell apoptosis triggered by CD95/Fas. On the basis of the suggested role of lipid rafts in conveying pro-apoptotic signals these results disclose new perspectives in the understanding of the mechanisms by which raft-mediated pro-apoptotic signals can directionally reach their target, i.e. the mitochondria, and trigger apoptosis execution.

  18. Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Murai Atsuko

    2012-07-01

    Full Text Available Abstract Background Canine hemangiosarcoma (HSA is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines. Results Seven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs, that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser473, mTOR complex 1 (mTORC1 at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1 at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines. Conclusions Our findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in

  19. Deriving the extinction to young stellar objects using [FeII] near-infrared emission lines. Prescriptions from GIANO high-resolution spectra

    CERN Document Server

    Pecchioli, Tommaso; Massi, Fabrizio; Oliva, Ernesto

    2016-01-01

    The near-infrared emission lines of Fe$^{+}$ at 1.257, 1.321, and 1.644 $\\mu$m share the same upper level; their ratios can then be exploited to derive the extinction to a line emitting region once the relevant spontaneous emission coefficients are known. This is commonly done, normally from low-resolution spectra, in observations of shocked gas from jets driven by Young Stellar Objects. In this paper we review this method, provide the relevant equations, and test it by analyzing high-resolution ($R \\sim 50000$) near-infrared spectra oftwo young stars, namely the Herbig Be star HD 200775 and the Be star V1478 Cyg, which exhibit intense emission lines. The spectra were obtained with the new GIANO echelle spectrograph at the Telescopio Nazionale Galileo. Notably, the high-resolution spectra allowed checking the effects of overlapping telluric absorption lines. A set of various determinations of the Einstein coefficients are compared to show how much the available computations affect extinction derivation. The m...

  20. Novel CMS lines in pigeonpea [Cajanus cajan (L. Millspaugh] derived from cytoplasmic substitutions, and their effective restoration and deployment in hybrid breeding

    Directory of Open Access Journals (Sweden)

    Abhishek Bohra

    2017-02-01

    Full Text Available The availability of stable cytoplasmic male sterile (CMS or A lines coupled with a robust restoration system (R lines is an essential prerequisite for efficient hybrid breeding. CMS-enabled hybrid technology holds immense potential to enhance the long-stagnant productivity of pigeonpea. In the present investigation, cytoplasmic substitutions were made in the nuclear backgrounds of early-maturing pigeonpea varieties or lines. Three new CMS lines (ICPL 88039A, Pusa 992A, and DPP 3-2A resulted from genetic crosses involving cytoplasmic donors from A2 (GT 288A and A4 (ICPA 2089 categories. In addition to visual inspection of anthers, pollen-staining techniques and scanning electron microscopy (SEM analysis were used to confirm pollen sterility. Further, given the relevance of the plant mitochondrial genome to CMS manifestation, 25 mitochondrion-specific DNA markers were assayed on these newly developed A lines and isogenic maintainer (B lines. DNA polymorphism between Pusa 992A and Pusa 992B as revealed by the nad7a_del marker confirmed the successful combination of sterilizing cytoplasm (A4 and nonrestoring nuclear background (Pusa 992. Such cytoplasm-specific DNA markers are required for A2-CMS as well. Further, to assess restoration ability, potential restorers were crossed with these CMS lines, and as a consequence, promising A × R combinations exhibiting 100% pollen fertility could be identified. In parallel, we also analyzed the inheritance patterns underlying fertility restoration using ICPL 88039A-derived F2 and BC1F1 populations, and established a monogenic dominant model to explain the phenomenon of A2-CMS restoration. In summary, we report the successful development of new CMS lines and describe their effective deployment in hybrid breeding of pigeonpea.

  1. Potential of on-line visible and near infrared spectroscopy for measurement of pH for deriving variable rate lime recommendations.

    Science.gov (United States)

    Tekin, Yücel; Kuang, Boyan; Mouazen, Abdul M

    2013-08-08

    This paper aims at exploring the potential of visible and near infrared (vis-NIR) spectroscopy for on-line measurement of soil pH, with the intention to produce variable rate lime recommendation maps. An on-line vis-NIR soil sensor set up to a frame was used in this study. Lime application maps, based on pH predicted by vis-NIR techniques, were compared with maps based on traditional lab-measured pH. The validation of the calibration model using off-line spectra provided excellent prediction accuracy of pH (R2 = 0.85, RMSEP = 0.18 and RPD = 2.52), as compared to very good accuracy obtained with the on-line measured spectra (R2 = 0.81, RMSEP = 0.20 and RPD = 2.14). On-line predicted pH of all points (e.g., 2,160) resulted in the largest overall field virtual lime requirement (1.404 t), as compared to those obtained with 16 validation points off-line prediction (0.28 t), on-line prediction (0.14 t) and laboratory reference measurement (0.48 t). The conclusion is that the vis-NIR spectroscopy can be successfully used for the prediction of soil pH and for deriving lime recommendations. The advantage of the on-line sensor over sampling with limited number of samples is that more detailed information about pH can be obtained, which is the reason for a higher but precise calculated lime recommendation rate.

  2. Signaling of glial cell line-derived neurotrophic factor and its receptor GFRα1 induce Nurr1 and Pitx3 to promote survival of grafted midbrain-derived neural stem cells in a rat model of Parkinson disease.

    Science.gov (United States)

    Lei, Zhinian; Jiang, Yu; Li, Tao; Zhu, Jianbao; Zeng, Shuilin

    2011-09-01

    Glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 have been implicated in the survival of ventral midbrain dopaminergic (DA) neurons, but the molecular mechanisms bywhich GDNF generates DA neurons in grafted midbrain-derived neural stem cells (mNSCs) are not understood. Midbrain-derived neural stem cells isolated from rat embryonic mesencephalon (embryonic day 12) were treated with GDNF or in combination with GFRα1 small interfering RNA. Reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used totest the expression of the orphan nuclear receptor Nurr1 and thetranscription factor Pitx3 and newborn tyrosine hydroxylase (TH)-positive cells. Treatment of mNSCs with GDNF increased mNSCs' sphere diameter, reduced expression of caspase 3, and increased expression of Bcl-2. Glial cell line-derived neurotrophic factor-treated mNSCs enhanced Nurr1 and Pitx3 expression and the fraction of TH-, TH/Pitx3-, and TH/Nurr1-positive cells in culture. Grafted GDNF-treated mNSCs significantly decreased apomorphine-induced rotation behavior in 6-hydroxydopamine-lesioned rats. Glialcell line-derived neurotrophic factor-treated mNSCs showed increased numbers of TH/Pitx3- and TH/Nurr1-postivie cells. The effect elicited by GDNF was inhibited by small interfering RNA-mediated knockdown of GFRα1. Our data demonstrate the contribution of GDNF to DA neuron development and may also elucidate pathogenetic mechanisms in Parkinson disease and contribute to the development of novel therapies for the disorder.

  3. SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

    Directory of Open Access Journals (Sweden)

    Zaborski Margarete

    2009-01-01

    Full Text Available Abstract Background SET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9(q34.11q34.13 has recently been described in T-cell acute lymphoblastic leukemia (T-ALL and in one case of acute myeloid leukemia (AML. The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene. Results Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Iβ-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH and array-based copy number analysis were both consistent with del(9(q34.11q34.13 as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein. Conclusion Cell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.

  4. Characterisation Of Forebrain Neurons Derived From Late-Onset Huntington’s Disease Human Embryonic Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Jonathan Christos Niclis

    2013-04-01

    Full Text Available Huntington's Disease (HD is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin gene. Recently, induced pluripotent stem cell lines carrying atypical and aggressive (CAG60+ HD variants have been generated, and perplexingly exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC lines carrying CAG37 and CAG51 repeats to assess whether typical late-onset expansions exhibit HD pathologies. HD hESC properties were assessed in comparison to wildtype control lines at undifferentiated states and throughout forebrain neuronal differentiation. Pluripotent HD lines demonstrate growth, viability, pluripotent gene expression, mitochondrial activity and forebrain specification that is indistinguishable from control lines. Expression profiles of crucial genes known to be dysregulated in HD remain unperturbed in the presence of mutant protein and throughout differentiation; however, elevated glutamate responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or differentiation mechanics but that neuronal progeny may possess the capacity to recapitulate neuropathologies seen in human patients. Such HD models will help further our understanding of the cascade of pathological events leading to disease onset and progression, while simultaneously facilitating the identification of candidate HD therapeutics.

  5. Epigenetic status of H19/IGF2 and SNRPN imprinted genes in aborted and successfully derived embryonic stem cell lines in non-human primates

    Directory of Open Access Journals (Sweden)

    Florence Wianny

    2016-05-01

    Full Text Available The imprinted genes of primate embryonic stem cells (ESCs often show altered DNA methylation. It is unknown whether these alterations emerge while deriving the ESCs. Here we studied the methylation patterns of two differentially methylated regions (DMRs, SNRPN and H19/IGF2 DMRs, during the derivation of monkey ESCs. We show that the SNRPN DMR is characteristically methylated at maternal alleles, whereas the H19/IGF2 DMR is globally highly methylated, with unusual methylation on the maternal alleles. These methylation patterns remain stable from the early stages of ESC derivation to late passages of monkey ESCs and following differentiation. Importantly, the methylation status of H19/IGF2 DMR and the expression levels of IGF2, H19, and DNMT3B mRNAs in early embryo-derived cells were correlated with their capacity to generate genuine ESC lines. Thus, we propose that these markers could be useful to predict the outcomes of establishing an ESC line in primates.

  6. Effects of hypoxia on expression of a panel of stem cell and chemosensitivity markers in glioblastoma cell line-derived spheroids

    DEFF Research Database (Denmark)

    Kolenda, Jesper; Jensen, Stine Skov; Aaberg-Jessen, Charlotte

    of a panel of stem cell and chemosensitivity markers was therefore investigated using glioma spheroids derived from the conventional glioblastoma cell line U87. The glioma spheroids were derived at normoxic (21% O2) and hypoxic (1% O2) culturing conditions in serum-free medium with EGF and bFGF. The entire......Glioblastomas are the most frequent and malignant primary brain tumor. Tumor stem cells in these tumors have recently been suggested to possess innate resistance mechanisms against radiation and chemotherapy possibly explaining their high level of therapeutic resistance. Moreover tumor hypoxia...... immunohistochemical panel included hypoxia (HIF-1α, HIF-2α), proliferation (Ki-67) and stem cell (CD133, nestin, podoplanin, Bmi-1, Sox-2) markers as well as markers related to chemosensitivity (MGMT, MDR-1, TIMP-1, Lamp-1). Since spheroids derived in hypoxia were smaller than in normoxia, a set of experiments...

  7. Derivation of the human induced pluripotent stem cell line MUi017-A from a patient with homozygous Hemoglobin Constant Spring

    Directory of Open Access Journals (Sweden)

    Wasinee Wongkummool

    2017-04-01

    Full Text Available Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52 year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.

  8. Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

    Directory of Open Access Journals (Sweden)

    Ana de la Cueva

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα, an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. METHODOLOGY/PRINCIPAL FINDINGS: ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS and thymidine kinase (TK1 levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. CONCLUSION/SIGNIFICANCE: Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

  9. Cell Line Derived 5-FU and Irinotecan Drug-Sensitivity Profiles Evaluated in Adjuvant Colon Cancer Trial Data

    DEFF Research Database (Denmark)

    Buhl, Ida Kappel; Gerster, Sarah; Delorenzi, Mauro

    2016-01-01

    patients who benefitted from the addition of irinotecan to 5-FU, we used gene expression profiles based on cell lines and clinical tumor material. These profiles were applied to expression data obtained from pretreatment formalin fixed paraffin embedded (FFPE) tumor tissue from 636 stage III colon cancer...

  10. Stiffness-Displacement Correlation from the RC Shear Wall Tests of the SAFE Program: Derivation of a Capacity Line Model

    Directory of Open Access Journals (Sweden)

    Francisco J. Molina

    2016-01-01

    Full Text Available The response of 13 reinforced concrete shear walls submitted to successive seismic tests has been postprocessed to produce time histories of secant stiffness and displacement oscillation amplitude. For every wall an envelope curve of displacement amplitude versus stiffness is identified which is fairly modelled by a straight line in double logarithmic scale. This relatively simple model, when used as a capacity line in combination with the demand response spectrum, is able to predict in an approximate manner the maximum response to the applied earthquakes. Moreover, the graphic representation of the demand spectrum and a unique model capacity line for a group of equal walls with different assumed design frequencies on them gives a visual interpretation of the different safety margins observed in the experiments for the respective walls. The same method allows as well constructing vulnerability curves for any design frequency or spectrum. Finally, the comparison of the different identified line models for the different walls allows us to assess the qualitative effect on the behaviour of parameters such as the reinforcement density or the added normal load.

  11. Derivation and long-term culture of an embryonic stem cell-like line from zebrafish blastomeres under feeder-free condition.

    Science.gov (United States)

    Ho, Sing Yee; Goh, Crystal Wei Pin; Gan, Jen Yang; Lee, Youn Sing; Lam, Millie Kuen Kuen; Hong, Ni; Hong, Yunhan; Chan, Woon Khiong; Shu-Chien, Alexander Chong

    2014-10-01

    Existing zebrafish embryonic stem (ES) cell lines are derived and maintained using feeder layers. We describe here the derivation and long-term culture of an ES cell-like line derived from zebrafish blastomeres without the use of feeder cells. This line, designated as ZES1, has been maintained for more than 800 days in defined Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, zebrafish embryo extract, trout serum, and human basic fibroblast growth factor. ZES1 cells possessed a morphology typical of ES cells, being round or polygonal in shape with a large nucleus and sparse cytoplasm and were mostly diploid. The cells formed individual colonies consisting of tightly packed cells that stained positively for alkaline phosphatase. ZES1 cells also formed embryoid bodies when transferred onto uncoated wells. The pluripotent nature of ZES1 cells was confirmed when they could be induced to differentiate in vitro into several cell types, through low- or high-density culture conditions. Treatment with retinoic acid also induced the differentiation of ZES1 cells into primarily neuronal cells. Using immunostaining and real-time polymerase chain reaction, we showed that Sox2, a known pluripotent marker in mammalian ES cells, was also present in ZES1 cells. Chimera experiments revealed that fluorescent-labeled ZES1 cells microinjected into zebrafish blastulas participated in the formation of all three germ layers. Using GFP-labeled ZES1 cells, chimera germline transmission was also demonstrated at the F1 generation. In conclusion, ZES1 cells possess both in vitro and in vivo pluripotency characteristics, indicating that nonmammalian ES cells can be readily derived and maintained for a long term under feeder-free culture conditions.

  12. Synergism of Cyclin-Dependent Kinase Inhibitors with Camptothecin Derivatives in Small Cell Lung Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Gerhard Hamilton

    2014-02-01

    Full Text Available Advanced small cell lung cancer (SCLC has a dismal prognosis. Modulation of the camptothecin topotecan, approved for second-line therapy, may improve response. Our recent finding of synergistic enhancement of the cytotoxic activity of camptothecin (CPT by cyclin-dependent kinase 4 inhibitors is extended here to a panel of camptothecin analogs comprising 10-hydroxy-CPT (HOCPT, topotecan (TPT; 9-[(dimethylamino-methyl]-10-hydroxy-CPT, 9-amino-CPT (9AC, 9-nitrocamptothecin (rubitecan, SN38 (7-ethyl-10-hydroxycamptothecin and 10-hydroxy-9-nitrocamptothecin (CPT109 in combination with PD0332991, CDK4I, roscovitine and olomoucine. SCLC cell lines employed are chemoresistant NCI-H417 and DMS153 and the chemosensitive SCLC26A line established at our institution. The CPT analogs exhibiting highest cytotoxicity towards the three SCLC lines tested were SN38 and 9AC, followed by rubitecan, HOCPT, TPT and CPT109. NCI-H417 and DMS153 revealed an approximately 25-fold and 7-fold higher resistance compared to the chemosensitive SCLC26A cell line. Whereas the CDK4/6 inhibitor PD0332991 proved less effective to chemosensitize SCLC cells to CPT analogs, the CDK inhibitors CDK4I, roscovitine and olomoucine gave comparable chemosensitization effects in combination with 9AC, SN38, rubitecan and to a lesser extent with TPT and CPT109, not directly related with topoisomerase mRNA expression. In conclusion, small chemical modifications of the parent CPT structure result in differing cytotoxicities and chemomodulatory effects in combination with CDKIs of the resulting analogs.

  13. Probing the mass-loss history of AGB and red supergiant stars from CO rotational line profiles - II. CO line survey of evolved stars: derivation of mass-loss rate formulae

    CERN Document Server

    De Beck, E; de Koter, A; Justtanont, K; Verhoelst, T; Kemper, F; Menten, K M M

    2010-01-01

    We aim to (1) set up simple and general analytical expressions to estimate mass-loss rates of evolved stars, and (2) from those calculate estimates for the mass-loss rates of asymptotic giant branch (AGB), red supergiant (RSG), and yellow hypergiant stars in our galactic sample. Rotationally excited lines of CO are a very robust diagnostic in the study of circumstellar envelopes (CSEs). When sampling different layers of the CSE, observations of these molecular lines lead to detailed profiles of kinetic temperature, expansion velocity, and density. A state-of-the-art, nonlocal thermal equilibrium, and co-moving frame radiative transfer code that predicts CO line intensities in the CSEs of late-type stars is used in deriving relations between stellar and molecular-line parameters, on the one hand, and mass-loss rate, on the other. We present analytical expressions for estimating the mass-loss rates of evolved stellar objects for 8 rotational transitions of the CO molecule, apply them to our extensive CO data se...

  14. Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines.

    Science.gov (United States)

    Somers-Edgar, Tiffany J; Taurin, Sebastien; Larsen, Lesley; Chandramouli, Anupama; Nelson, Mark A; Rosengren, Rhonda J

    2011-02-01

    Estrogen receptor (ER)-negative breast cancer is an aggressive form that currently requires more drug treatment options. Thus, we have further modified cyclohexanone derivatives of curcumin and examined them for cytotoxicity towards ER-negative human breast cancer cells. Two of the analogs screened elicited increased cytotoxic potency compared to curcumin and other previously studied derivatives. Specifically, 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) elicited EC(50) values of 1.54 and 1.10 µM, respectively, in MDA-MB-231 cells and EC(50) values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC(50) values of 7.6 and 2.4 µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G(2)/M-phase cell cycle arrest and apoptosis. RL90 and RL91 also modulated the expression of key cell signaling proteins, specifically, in SKBr3 cells, protein levels of Her-2, Akt, and NFκB were decreased in a time-dependent manner, while activity of stress kinases JNK1/2 and P38 MAPK were increased. Signaling events in MDA-MB-231 cells were differently implicated, as EGFR protein levels were decreased and activity of GSK-3β transiently decreased, while β-catenin protein level and activity of P38 MAPK, Akt, and JNK1/2 were transiently increased. In conclusion replacement of the phenyl group of cyclohexanone derived curcumin derivatives with heterocyclic rings forms a class of second-generation analogs that are more potent than both curcumin and other derivatives. These new derivatives provide a platform for the further development of drugs for the treatment of ER-negative breast cancer.

  15. Low or undetectable TPO receptor expression in malignant tissue and cell lines derived from breast, lung, and ovarian tumors

    Directory of Open Access Journals (Sweden)

    Erickson-Miller Connie L

    2012-09-01

    Full Text Available Abstract Background Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues. Methods Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR, and for TPO-R protein expression by immunohistochemistry (IHC. Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot. Results MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43% and malignant (3-17% breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines. Conclusions Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and

  16. Inhibition spectrum studies of microthecin and other anhydrofructose derivatives using selected strains of Gram-positive and –negative bacteria, yeast and moulds, and investigation of the cytotoxicity of microthecin to malignant blood cell lines

    DEFF Research Database (Denmark)

    Fiskesund, R.; Thomas, L.V.; Schobert, M.

    2009-01-01

    Aims: To prepare 1,5-anhydro-d-fructose (AF) derivatives, test their microbial inhibition spectrum, and to further examine the most effective AF derivative against Pseudomonas aeruginosa and malignant blood cell lines. Methods and Results: Microthecin and nine other AF derivatives were synthesize...

  17. A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure

    Institute of Scientific and Technical Information of China (English)

    Ge Lin; Qi OuYang; Xiaoying Zhou; Yifan Gu; Ding Yuan; Wen Li; Gang Liu; Tiancheng Liu; Guanexiu Lu

    2007-01-01

    Homozygous human embryonic stem cells (hESCs) are thought to be better cell sources for hESC banking because their human leukocyte antigen (HLA) haplotype would strongly increase the degree of matching for certain populations with relatively smaller cohorts of cell lines. Homozygous hESCs can be generated from parthenogenetic embryos, but only heterozygous hESCs have been established using the current strategy to artificially activate the oocyte without second polar body extrusion. Here we report the first successful derivation of a human homozygous ESC line (chHES-32) from a one-pronuclear oocyte following routine in vitro fertilization treatment. cAHES-32 cells express common markers and genes with normal hESCs. They have been propagated in an undifferentiated state for more than a year (>P50) and have maintained a stable karyotype of 46, XX. When differentiated in vivo and in vitro, c/zHES-32 cells can form derivatives from all three embryonic germ layers. The almost undetectable expression of five paternally expressed imprinted genes and their HLA genotype identical to the oocyte donor indicated their parthenogenetic origin. Using genome-wide single-nucleotide polymorphism analysis and DNA fingerprinting, the homozygosity of c/zHES-32 cells was further confirmed. The results indicated that 'unwanted' one-pronuclear oocytes might be a potential source for human homozygous and parthenogenetic ESCs, and suggested an alternative strategy for obtaining homozygous hESC lines from parthenogenetic haploid oocytes.

  18. Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas

    Science.gov (United States)

    Lu, Y.; Aguirre, A.A.; Work, T.M.; Balazs, G.H.; Nerurkar, V.R.; Yanagihara, R.

    2000-01-01

    Serial cultivation of cell lines derived from lung, testis, periorbital and tumor tissues of a green turtle (Chelonia mydas) with fibropapillomas resulted in the in vitro formation of tumor-like cell aggregates, ranging in size from 0.5 to 2.0 mm in diameter. Successful induction of tumor-like aggregates was achieved in a cell line derived from lung tissue of healthy green turtles, following inoculation with cell-free media from these tumor-bearing cell lines, suggesting the presence of a transmissible agent. Thin-section electron microscopy of the cell aggregates revealed massive collagen deposits and intranuclear naked viral particles, measuring 50??5 nm in diameter. These findings, together with the morphological similarity between these tumor-like cell aggregates and the naturally occurring tumor, suggest a possible association between this novel virus and the disease. Further characterization of this small naked virus will clarify its role in etiology of green turtle fibropapilloma, a life-threatening disease of this endangered marine species. Copyright (C) 2000 Elsevier Science B.V.

  19. Conditioned Media from Human Adipose Tissue-Derived Mesenchymal Stem Cells and Umbilical Cord-Derived Mesenchymal Stem Cells Efficiently Induced the Apoptosis and Differentiation in Human Glioma Cell Lines In Vitro

    Directory of Open Access Journals (Sweden)

    Chao Yang

    2014-01-01

    Full Text Available Human mesenchymal stem cells (MSCs have an intrinsic property for homing towards tumor sites and can be used as tumor-tropic vectors for tumor therapy. But very limited studies investigated the antitumor properties of MSCs themselves. In this study we investigated the antiglioma properties of two easily accessible MSCs, namely, human adipose tissue-derived mesenchymal stem cells (ASCs and umbilical cord-derived mesenchymal stem cells (UC-MSCs. We found (1 MSC conditioned media can significantly inhibit the growth of human U251 glioma cell line; (2 MSC conditioned media can significantly induce apoptosis in human U251 cell line; (3 real-time PCR experiments showed significant upregulation of apoptotic genes of both caspase-3 and caspase-9 and significant downregulation of antiapoptotic genes such as survivin and XIAP after MSC conditioned media induction in U 251 cells; (4 furthermore, MSCs conditioned media culture induced rapid and complete differentiation in U251 cells. These results indicate MSCs can efficiently induce both apoptosis and differentiation in U251 human glioma cell line. Whereas UC-MSCs are more efficient for apoptosis induction than ASCs, their capability of differentiation induction is not distinguishable from each other. Our findings suggest MSCs themselves have favorable antitumor characteristics and should be further explored in future glioma therapy.

  20. In vitro priming of tumor-specific cytotoxic T lymphocytes using allogeneic dendritic cells derived from the human MUTZ-3 cell line.

    Science.gov (United States)

    Santegoets, Saskia J A M; Schreurs, Marco W J; Masterson, Allan J; Liu, Ying Poi; Goletz, Steffen; Baumeister, Hans; Kueter, Esther W M; Lougheed, Sinéad M; van den Eertwegh, Alfons J M; Scheper, Rik J; Hooijberg, Erik; de Gruijl, Tanja D

    2006-12-01

    The adoptive transfer of in vitro-induced and expanded tumor-specific cytotoxic T lymphocytes (CTL) presents a promising immunotherapeutic approach for the treatment of cancer. The in vitro induction of tumor-reactive CTL requires repeated stimulation of CTL precursors with dendritic cells (DC). To circumvent problems like scarcity of blood DC precursors and donor variability, it would be attractive to use DC from a non-autologous, unlimited source. DCs derived from the human acute myeloid leukemia (AML) cell line MUTZ-3 are attractive candidates since these DCs closely resemble monocyte-derived DC (MoDC) in terms of phenotype and T cell stimulatory capacity. Here we demonstrate that functional CTL clones could be generated against multiple tumor-associated antigens, i.e., human telomerase reverse transcriptase (hTERT), ErbB3-binding protein-1 (Ebp1), carcinoembryonic antigen (CEA) and Her-2/neu, by stimulating CD8beta(+) CTL precursors with peptide-loaded allogeneic, HLA-A2-matched MUTZ-3-derived DC. A consistent induction capacity, as determined by MHC tetramer-binding, was found in multiple donors and comparable to autologous peptide-loaded MoDC. Functional characterization at the clonal level revealed the priming of CTL that recognized endogenously processed epitopes on tumor cell lines in an HLA-A2-restricted fashion. Our data indicate that MUTZ-3-derived DC can be used as stimulator cells for in vitro priming and expansion of functional TAA-specific effector CTL. MUTZ-3-derived DCs thus represent a ready and standardized source of allogeneic DC to generate CTL for therapeutic adoptive transfer strategies.

  1. Molecular dissection of Tomato leaf curl virus resistance in tomato line TY172 derived from Solanum peruvianum.

    Science.gov (United States)

    Anbinder, Ilana; Reuveni, Moshe; Azari, Raviv; Paran, Ilan; Nahon, Sahadia; Shlomo, Haviva; Chen, Lea; Lapidot, Moshe; Levin, Ilan

    2009-08-01

    Tomato yellow leaf curl virus (TYLCV) is devastating to tomato (Solanum lycopersicum) crops and resistant cultivars are highly effective in controlling the disease. The breeding line TY172, originating from Solanum peruvianum, is highly resistant to TYLCV. To map quantitative trait loci (QTLs) controlling TYLCV resistance in TY172, appropriate segregating populations were analyzed using 69 polymorphic DNA markers spanning the entire tomato genome. Results show that TYLCV resistance in TY172 is controlled by a previously unknown major QTL, originating from the resistant line, and four additional minor QTLs. The major QTL, we term Ty-5, maps to chromosome 4 and accounts for 39.7-46.6% of the variation in symptom severity among segregating plants (LOD score 33-35). The minor QTLs, originated either from the resistant or susceptible parents, were mapped to chromosomes 1, 7, 9 and 11, and contributed 12% to the variation in symptom severity in addition to Ty-5.

  2. A CLONALLY DERIVED CELL LINE,9L-EGFR IS USEFUL FOR THE STUDIES OF CANCER CELLS BEARING EGF RECEPTOR

    Institute of Scientific and Technical Information of China (English)

    Lin Qi; Rajesh Agarwal; Rana Singh; Gail S. Harrisona; L.Michael Glodea

    2003-01-01

    Since the epidermal growth factor receptor (EGFR) is a key regulator in cell signaling pathways of cancer cell. To investigate the mechanism between cancer cells survival and its EGFR expression, drug selection of cancer cells target therapy, we generated a cell line, 9L-EGFR, which stably expressed human EGFR; the parental rat glioma cell line, 9L, does not contain endogenous EGFR message or protein. Our results show that 9L-EGFR cells had high levels of EGFR on their cell surface by using RT-PCR, Western analysis and Flow cytometry analysis. The EGFR transfected into 9L cells was capable of being activated by EGF, in which either phosphorylated (p-EGFR) or total (EGFR) was showed by Western blot. This investigation may contribute to the further studies of cancer cells bearing EGFR.

  3. MSLN Gene Silencing Has an Anti-Malignant Effect on Cell Lines Overexpressing Mesothelin Deriving from Malignant Pleural Mesothelioma

    Science.gov (United States)

    Melaiu, Ombretta; Stebbing, Justin; Lombardo, Ylenia; Bracci, Elisa; Uehara, Norihisa; Bonotti, Alessandra; Cristaudo, Alfonso; Foddis, Rudy; Mutti, Luciano; Barale, Roberto; Gemignani, Federica

    2014-01-01

    Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. So far, mesothelin (MSLN) has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373) has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e. NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A). MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA) to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer. PMID:24465798

  4. MSLN gene silencing has an anti-malignant effect on cell lines overexpressing mesothelin deriving from malignant pleural mesothelioma.

    Directory of Open Access Journals (Sweden)

    Ombretta Melaiu

    Full Text Available Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM are still poorly characterized. So far, mesothelin (MSLN has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373 has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e., NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A. MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer.

  5. Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.B11

    DEFF Research Database (Denmark)

    Hansen, Susanne Kofoed; Borland, Helena; Hasholt, Lis Frydenreich;

    2016-01-01

    Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by an expansion of the CAG-repeat in ATXN3. In this study, induced pluripotent stem cells (iPSCs) were generated from SCA3 patient dermal fibroblasts by electroporation with episomal plasmids encoding L......PSC line could be a useful tool for the investigation of SCA3 disease mechanisms....

  6. Generation and Characterization of Vascular Smooth Muscle Cell Lines Derived from a Patient with a Bicuspid Aortic Valve

    Directory of Open Access Journals (Sweden)

    Pamela Lazar-Karsten

    2016-04-01

    Full Text Available Thoracic aortic dilation is the most common malformation of the proximal aorta and is responsible for 1%–2% of all deaths in industrialized countries. In approximately 50% of patients with a bicuspid aortic valve (BAV, dilation of any or all segments of the aorta occurs. BAV patients with aortic dilation show an increased incidence of cultured vascular smooth muscle cell (VSMC loss. In this study, VSMC, isolated from the ascending aorta of BAV, was treated with Simian virus 40 to generate a BAV-originated VSMC cell line. To exclude any genomic DNA or cross-contamination, highly polymorphic short tandem repeats of the cells were profiled. The cells were then characterized using flow cytometry and karyotyping. The WG-59 cell line created is the first reported VSMC cell line isolated from a BAV patient. Using an RT2 Profiler PCR Array, genes within the TGFβ/BMP family that are dependent on losartan treatment were identified. Endoglin was found to be among the regulated genes and was downregulated in WG-59 cells following treatment with different losartan concentrations, when compared to untreated WG-59 cells.

  7. Apoptosis Activation in Human Lung Cancer Cell Lines by a Novel Synthetic Peptide Derived from Conus californicus Venom

    Directory of Open Access Journals (Sweden)

    Irasema Oroz-Parra

    2016-02-01

    Full Text Available Lung cancer is one of the most common types of cancer in men and women and a leading cause of death worldwide resulting in more than one million deaths per year. The venom of marine snails Conus contains up to 200 pharmacologically active compounds that target several receptors in the cell membrane. Due to their diversity and specific binding properties, Conus toxins hold great potential as source of new drugs against cancer. We analyzed the cytotoxic effect of a 17-amino acid synthetic peptide (s-cal14.1a that is based on a native toxin (cal14.1a isolated from the sea snail Conus californicus. Cytotoxicity studies in four lung cancer cell lines were complemented with measurement of gene expression of apoptosis-related proteins Bcl-2, BAX and the pro-survival proteins NFκB-1 and COX-2, as well as quantification of caspase activity. Our results showed that H1299 and H1437 cell lines treated with s-call4.1a had decreased cell viability, activated caspases, and reduced expression of the pro-survival protein NFκB-1. To our knowledge, this is the first report describing activation of apoptosis in human lung cancer cell lines by s-cal14.1a and we offer insight into the possible mechanism of action.

  8. Characterization of two novel cell lines with distinct heterogeneity derived from a single human bile duct carcinoma.

    Directory of Open Access Journals (Sweden)

    Jinghan Wang

    Full Text Available BACKGROUND: Intratumoral heterogeneity reflects subclonal diversity and accounts for a variety of clinically defined phenotypes including the development of drug resistance and recurrence. However, intratumoral heterogeneity of bile duct carcinoma (BDC is rarely studied. METHODS: Two highly heterogeneous cell lines named EH-CA1a and EH-CA1b were established from a primary tumor tissue of a pathologically proven BDC. Distinct heterogeneity and underlying mechanisms of two cell lines in karyotype, colony formation, tumorgenicity, and sensitivity to chemoradiotherapy were intensively studied. RESULTS: Both cell lines showed typical morphology of cancer cells. EH-CA1a cells grew as free-floating aggregates, while EH-CA1b cells grew adherently as a monolayer. EH-CA1a cells had higher cloning efficiencies and were able to keep proliferating under hypoxic condition. Coincidentally, hypoxia-induced factor-1α (HIF1α and vascular endothelial growth factor (VEGF mRNA were significantly higher in EH-CA1a cells than in EH-CA1b cells. Both cell lines were tumorigenic in nude mouse, however, EH-CA1a cells showed more aggressive characteristics. Most importantly, the EH-CA1a cells showed much more resistance against radiation and chemotherapy with gemcitabine. Metastasis-related genes including matrix metalloproteinase 2 (MMP-2, MMP-9, epithelial-mesenchymal transition (EMT markers such as Vimentin, Snail, and Twist, are more highly expressed in EH-CA1a cells than in EH-CA1b cells. Moreover, the percentage of cells expressing cancer stem cell-like marker, CD133, in EH-CA1a cells is much higher than that in EH-CA1b cells. Moreover, knockdown of CD133 in both EH-CA1a and EH-CA1b cells significantly reduced their invasive potential and increased their sensitivities to radiation and gemcitabine, suggesting the differential expression of CD133 protein may partially account for the difference in malignancy between these two cancer cells. CONCLUSION: Establishment

  9. A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo

    Science.gov (United States)

    Fonseca, Simone Aparecida Siqueira; Costas, Roberta Montero; Morato-Marques, Mariana; Costa, Silvia; Alegretti, Jose Roberto; Rosenberg, Carla; da Motta, Eduardo Leme Alves; Serafini, Paulo C.; Pereira, Lygia V.

    2015-01-01

    Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1–2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo´s missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage. PMID:26540511

  10. Serial enrichment of spermatogonial stem and progenitor cells (SSCs) in culture for derivation of long-term adult mouse SSC lines.

    Science.gov (United States)

    Martin, Laura A; Seandel, Marco

    2013-02-25

    Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells(1-3). Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines(4). However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state. Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors(5,6). Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies(7). Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types(8). Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC(2

  11. Accelerated Senescence and Enhanced Disease Resistance in Hybrid Chlorosis Lines Derived from Interspecific Crosses between Tetraploid Wheat and Aegilops tauschii

    OpenAIRE

    Hiroki Nakano; Nobuyuki Mizuno; Yukio Tosa; Kentaro Yoshida; Pyoyun Park; Shigeo Takumi

    2015-01-01

    Hybrid chlorosis, a type of hybrid incompatibility, has frequently been reported in inter- and intraspecific crosses of allopolyploid wheat. In a previous study, we reported some types of growth abnormalities such as hybrid necrosis and observed hybrid chlorosis with mild or severe abnormalities in wheat triploids obtained in crosses between tetraploid wheat cultivar Langdon and four Ae. tauschii accessions and in their derived synthetic hexaploids. However, the molecular mechanisms underlyin...

  12. Cancer stem-like sphere cells induced from de-differentiated hepatocellular carcinoma-derived cell lines possess the resistance to anti-cancer drugs.

    Science.gov (United States)

    Hashimoto, Noriaki; Tsunedomi, Ryouichi; Yoshimura, Kiyoshi; Watanabe, Yusaku; Hazama, Shoichi; Oka, Masaaki

    2014-09-27

    Cancer stem cells (CSCs) are thought to play important roles in therapy-resistance. In this study, we induced cancer stem-like cells from hepatocellular carcinoma (HCC) cell lines using a unique medium, and examined their potential for resistance to anti-cancer drugs. The human HCC cell lines SK-HEP-1 (SK), HLE, Hep 3B, and HuH-7 were used to induce cancer stem-like cells with our sphere induction medium supplemented with neural survival factor-1. NANOG and LIN28A were examined as stemness markers. Several surface markers for CSC such as CD24, CD44, CD44 variant, and CD90 were analyzed by flow-cytometry. To assess the resistance to anti-cancer drugs, the MTS assay, cell cycle analysis, and reactive oxygen species (ROS) activity assay were performed. Poorly differentiated HCC derived SK and undifferentiated HCC derived HLE cell lines efficiently formed spheres of cells (SK-sphere and HLE-sphere), but well-differentiated HCC-derived HuH-7 and Hep 3B cells did not. SK-spheres showed increased NANOG, LIN28A, and ALDH1A1 mRNA levels compared to parental cells. We observed more CD44 variant-positive cells in SK-spheres than in parental cells. The cell viability of SK-spheres was significantly higher than that of SK cells in the presence of several anti-cancer drugs except sorafenib (1.7- to 7.3-fold, each P < 0.05). The cell cycle of SK-spheres was arrested at the G0/G1 phase compared to SK cells. SK-spheres showed higher ABCG2 and HIF1A mRNA expression and lower ROS production compared to parental cells. Our novel method successfully induced cancer stem-like cells, which possessed chemoresistance that was related to the cell cycle, drug efflux, and ROS.

  13. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

    Directory of Open Access Journals (Sweden)

    Coppock Donald L

    2008-12-01

    Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

  14. Preliminary evaluation of the toxicity of some synthetic furan derivatives in two cell lines and Artemia salina.

    Science.gov (United States)

    Amaro, María I; Monasterios, Melina; Avendaño, Milagros; Charris, Jaime

    2009-01-01

    This study describes the preliminary toxicity evaluation of five new furan derivatives, 2-[2-acetylamino-2-[(benzothiazolyl-substituted)aminocarbonyl]vinyl]-5-nitro furane (compounds A, B, D and E) and 2-[2-phenylamino-2-[benzothiazolylaminocarbonyl]vinyl]furane (compound C). Cytotoxicity was determined using the MTT (tetrazolium salt) method over BHK21 (Syrian baby hamster kidney) and Hep-2 (human larynx carcinoma) cells, which had previously been used to evaluate the cytotoxicity of the 5-nitrofuran derivatives. The lethal concentration 50 (LC(50)) was determined using brine shrimp (Artemia salina) bioassay. Nitrofurantoin was used as reference compound. The results demonstrate that BHK21 cells are more sensitive than Hep-2 cells. This structurally related serial of compounds shows a differential toxicity, which is an indication that the toxicity naturally arising from the nitro group can be modulated by the substituents over the furan ring. Additionally, compound C, the only derivative with no nitro group, was least toxic to Hep-2, but exhibits toxicity to BHK21 cells and brine shrimp. The LC(50 )brine shrimp test (BST) bioassay results were as follows: A, 654.2 microg ml(-1); B, 50.0 microg ml(-1); C, 533.4 microg ml(-1); D, 172.1 microg ml(-1); E, 76.4 microg ml(-1), and NF, >1000 microg ml(-1).

  15. Evaluation of the Cytotoxicity of α-Cyclodextrin Derivatives on the Caco-2 Cell Line and Human Erythrocytes

    Directory of Open Access Journals (Sweden)

    Eszter Róka

    2015-11-01

    Full Text Available Cyclodextrins, even the 6-membered α-cyclodextrin, are approved in the various pharmacopoeias as pharmaceutical excipients for solubilizing and stabilizing drugs as well as for controlling drug release. Recently α-cyclodextrin has also been marketed as health food with beneficial effects on blood lipid profiles. However, the concentration of α-cyclodextrin used may be very high in these cases, and its toxic attributes have to be seriously considered. The objective of this study was to investigate the cytotoxicity of various, differently substituted α-cyclodextrin derivatives and determine relationship between the structures and cytotoxicity. Three different methods were used, viability tests (MTT assay and Real Time Cell Electronic Sensing on Caco-2 cells as well as hemolysis test on human red blood cells. The effect of α-cyclodextrin derivatives resulted in concentration-dependent cytotoxicity, so the IC50 values have been determined. Based on our evaluation, the Real Time Cell Electronic Sensing method is the most accurate for describing the time and concentration dependency of the observed toxic effects. Regarding the cytotoxicity on Caco-2 cells, phosphatidylcholine extraction may play a main role in the mechanism. Our results should provide help in selecting those α-cyclodextrin derivatives which have the potential of being used safely in medical formulations.

  16. Induction of apoptosis in the human promyelocytic leukemia cell line HL60 by falconensone A and its derivatives, new polyenes.

    Science.gov (United States)

    Takahashi, N; Kubo, Y; Iwahori, A; Kawai, K I; Fukui, T

    2000-06-01

    Falconensones A and B are a new type of yellow pigment with structural similarity to retinoic acid isolated from the mycelial extract of ascomycetous fungi, Emericella falconensis or Emericella fruticulosa. In the present study we show that falconensone A alone induced apoptosis of HL60 human leukemia cells, while falconensone B, the 4'-nor-methyl derivative of falconensone A, had much lower activity. The synthetic derivatives of falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime, were more potent than natural falconensone A and B as far as the induction of apoptosis was concerned. The induction of apoptosis by the falconensones correlated with their inhibition of cell growth. In addition, falconensones A and B, and falconensone A dioxime, increased the generation of intracellular reactive oxygen species, while falconensone A p-bromophenylhydrazone was inactive. These results suggest that falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime are potential new apoptosis-inducing agents. The enhanced generation of reactive oxygen species in cells may be involved in apoptosis induced by falconensone A and falconensone A dioxime, but not by falconensone A p-bromophenylhydrazone. It is also suggested that the methyl residue at the 4' position of the falconensone A cyclopentenone ring may be essential for the induction of apoptosis. Based on these results, falconensone A and its derivatives may be clinically useful in the treatment of some leukemias.

  17. Insulin-like growth factors and the multiplication of Tera-2, a human teratoma-derived cell line.

    Science.gov (United States)

    Biddle, C; Li, C H; Schofield, P N; Tate, V E; Hopkins, B; Engstrom, W; Huskisson, N S; Graham, C F

    1988-07-01

    A human teratoma cell line (Tera-2) was grown in serum-free medium, and the population multiplication was stimulated by the addition of somatomedins/insulin-like growth factors (IGFs). Both IGF-I and IGF-II gave maximal stimulation when added daily at 10 ng ml-1. The IGFs did not substantially change the labelling index of the cells, and the IGFs appeared to exert their effect on population multiplication by increasing cell survival. Membranes isolated from Tera-2 cells displayed both type 1 and type 2 IGF receptors.

  18. Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.A11

    DEFF Research Database (Denmark)

    Hansen, Susanne Kofoed; Borland, Helena; Hasholt, Lis Frydenreich;

    2016-01-01

    Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by a CAG-repeat expanding mutation in ATXN3. We generated induced pluripotent stem cells (iPSCs) from a SCA3 patient by electroporation of dermal fibroblasts with episomal plasmids encoding L-MYC, LIN28......, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of genomically integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. This iPSC line...

  19. Ukrain(TM), a semisynthetic Chelidonium majus alkaloid derivative, acts by inhibition of tubulin polymerization in normal and malignant cell lines.

    Science.gov (United States)

    Panzer, A; Hamel, E; Joubert, A M; Bianchi, P C; Seegers, J C

    2000-11-28

    Ukrain(TM) has been described as a semisynthetic Chelidonium majus alkaloid derivative, which exhibits selective toxicity towards malignant cells only. Its mechanism of action has hitherto been uncertain. We found that Ukrain(TM) inhibits tubulin polymerization, leading to impaired microtubule dynamics. This results in activation of the spindle checkpoint and thus a metaphase block. The effects of Ukrain(TM) on the growth, cell cycle progression and morphology of two normal, two transformed and two malignant cell lines did not differ. We could thus find no evidence for the selective cytotoxicity previously reported for Ukrain(TM).

  20. The effect of the two epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) on cell lines established from patients with small cell carcinoma of the lung

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, Lars; Christensen, I J;

    1987-01-01

    To determine whether there is any difference between the two epipodophyllotoxin derivatives etoposide and teniposide in their therapeutic effect in small cell carcinoma of the lung (SCCL), they were compared against five human SCCL cell lines in vitro. When the two were compared at equimolar...... is not accompanied by an equivalent increase in toxicity. The concentrations used for the 1-h incubation were about 100-fold the concentrations used in the experiments with continuous incubation to obtain the same degree of cell kill for both drugs. This suggests that they should be given according to a continuous...

  1. QTL mapping of starch granule size in common wheat using recombinant inbred lines derived from a PH82-2/Neixiang 188 cross

    OpenAIRE

    Nan Feng; Zhonghu He; Yong Zhang; Xianchun Xia; Yan Zhang

    2013-01-01

    Starch is a crucial component determining the processing quality of wheat (Triticum aestivum L.)-based products. Wheat starch generally contains A-type and B-type starch granules, having different effects on starch properties and end-use qualities. In the present study, 240 recombinant inbred lines (RILs) derived from a PH82-2/Neixiang 188 cross were grown in Anyang, Henan, China, during three cropping seasons. A-type and B-type granule contents were determined using a laser diffraction parti...

  2. The roles of glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor and nerve growth factor during the final stage of folliculogenesis: a focus on oocyte maturation.

    Science.gov (United States)

    Linher-Melville, Katja; Li, Julang

    2013-02-01

    Neurotrophic factors were first identified to promote the growth, survival or differentiation of neurons and have also been associated with the early stages of ovarian folliculogenesis. More recently, their effects on the final stage of follicular development, including oocyte maturation and early embryonic development, have been reported. Glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which are expressed in numerous peripheral tissues outside of the CNS, most notably the ovary, are now known to stimulate oocyte maturation in various species, also enhancing developmental competence. The mechanisms that underlie their actions in antral follicles, as well as the targets ultimately controlled by these factors, are beginning to emerge. GDNF, BDNF and NGF, alone or in combination, could be added to the media currently utilized for in vitro oocyte maturation, thereby potentially increasing the production and/or quality of early embryos.

  3. Impact of 1.8-GHz radiofrequency radiation (RFR) on DNA damage and repair induced by doxorubicin in human B-cell lymphoblastoid cells.

    Science.gov (United States)

    Zhijian, Chen; Xiaoxue, Li; Yezhen, Lu; Shijie, Chen; Lifen, Jin; Jianlin, Lou; Deqiang, Lu; Jiliang, He

    2010-01-01

    In the present in vitro study, a comet assay was used to determine whether 1.8-GHz radiofrequency radiation (RFR, SAR of 2W/kg) can influence DNA repair in human B-cell lymphoblastoid cells exposed to doxorubicin (DOX) at the doses of 0microg/ml, 0.05microg/ml, 0.075microg/ml, 0.10microg/ml, 0.15microg/ml and 0.20microg/ml. The combinative exposures to RFR with DOX were divided into five categories. DNA damage was detected at 0h, 6h, 12h, 18h and 24h after exposure to DOX via the comet assay, and the percent of DNA in the tail (% tail DNA) served as the indicator of DNA damage. The results demonstrated that (1) RFR could not directly induce DNA damage of human B-cell lymphoblastoid cells; (2) DOX could significantly induce DNA damage of human B-cell lymphoblastoid cells with the dose-effect relationship, and there were special repair characteristics of DNA damage induced by DOX; (3) E-E-E type (exposure to RFR for 2h, then simultaneous exposure to RFR and DOX, and exposure to RFR for 6h, 12h, 18h and 24h after exposure to DOX) combinative exposure could obviously influence DNA repair at 6h and 12h after exposure to DOX for four DOX doses (0.075microg/ml, 0.10microg/ml, 0.15microg/ml and 0.20microg/ml) in human B-cell lymphoblastoid cells. Copyright 2009 Elsevier B.V. All rights reserved.

  4. Derivation, characterization and differentiation of a new human embryonic stem cell line from a Chinese hatched blastocyst assisted by a non-contact laser system.

    Science.gov (United States)

    Wu, Rongrong; Xu, Chenming; Jin, Fan; Tan, Zhou; Gu, Bin; Chen, Liangbiao; Yao, Xing; Zhang, Ming

    2010-08-01

    Currently worldwide attention has focused on the derivation of human embryonic stem cells (hESCs) for future therapeutic medicine. However, the majority of existing hESCs are directly or indirectly exposed to non-human materials during their derivation and/or propagation, which greatly restrict their therapeutic potential. Besides the efforts to improve culture systems, the derivation procedure, especially blastocyst manipulation, needs to be optimized. We adopted a non-contact laser-assisted hatching system in combination with sequential culture process to obtain hatched blastocysts as materials for hESC derivation, and derived a hESC line ZJUhES-1 of a Chinese population without exposure to any non-human materials during blastocyst manipulation. ZJUhES-1 satisfies the criteria of pluripotent hESCs: typically morphological characteristics; the expression of alkaline phosphatase, human telomerase reverse transcriptase and multiple hESC-specific markers including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4, Nanog, Rex-1, Sox-2, UTF-1, Connexins 43 and 45, TERF-1 and TERF-2, Glut-1, BCRP-1/ABCG-2, GDF3, LIN28, FGF4, Thy-1, Cripto1/TDGF1, AC133 as well as SMAD1/2/3/5; extended proliferative capacity; maintenance of a stable male karyotype after long-term cultivation; and robust multiple-lineage developmental potentials both in vivo and in vitro. Moreover, ZJUhES-1 has distinct identity revealed from DNA fingerprinting. Our xeno-free blastocyst manipulation procedure may promote the progression toward clinical-grade hESC derivation.

  5. Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

    DEFF Research Database (Denmark)

    Bauer, Matthias; Meyer, Morten; Brevig, Thomas

    2002-01-01

    Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM...... tissue grafts to PD patients indicate that one reason for the poor outcome of neural transplantation is the low survival and differentiation of grafted dopaminergic neurons. To improve dopaminergic cell survival through a gene-therapeutic approach we have established and report here results of lipid...

  6. A novel cell line derived from pleomorphic adenoma expresses MMP2, MMP9, TIMP1, TIMP2, and shows numeric chromosomal anomalies.

    Directory of Open Access Journals (Sweden)

    Aline Semblano Carreira Falcão

    Full Text Available Pleomorphic adenoma is the most common salivary gland neoplasm, and it can be locally invasive, despite its slow growth. This study aimed to establish a novel cell line (AP-1 derived from a human pleomorphic adenoma sample to better understand local invasiveness of this tumor. AP-1 cell line was characterized by cell growth analysis, expression of epithelial and myoepithelial markers by immunofluorescence, electron microscopy, 3D cell culture assays, cytogenetic features and transcriptomic study. Expression of matrix metalloproteinases (MMPs and their tissue inhibitors (TIMPs was also analyzed by immunofluorescence and zymography. Furthermore, epithelial and myoepithelial markers, MMPs and TIMPs were studied in the tumor that originated the cell line. AP-1 cells showed neoplastic epithelial and myoepithelial markers, such as cytokeratins, vimentin, S100 protein and smooth-muscle actin. These molecules were also found in vivo, in the tumor that originated the cell line. MMPs and TIMPs were observed in vivo and in AP-1 cells. Growth curve showed that AP-1 exhibited a doubling time of 3.342 days. AP-1 cells grown inside Matrigel recapitulated tumor architecture. Different numerical and structural chromosomal anomalies were visualized in cytogenetic analysis. Transcriptomic analysis addressed expression of 7 target genes (VIM, TIMP2, MMP2, MMP9, TIMP1, ACTA2 e PLAG1. Results were compared to transcriptomic profile of non-neoplastic salivary gland cells (HSG. Only MMP9 was not expressed in both libraries, and VIM was expressed solely in AP-1 library. The major difference regarding gene expression level between AP-1 and HSG samples occurred for MMP2. This gene was 184 times more expressed in AP-1 cells. Our findings suggest that AP-1 cell line could be a useful model for further studies on pleomorphic adenoma biology.

  7. Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots.

    Science.gov (United States)

    Kung, Yi-Jung; Yu, Tsong-Ann; Huang, Chiung-Huei; Wang, Hui-Chin; Wang, Shin-Lan; Yeh, Shyi-Dong

    2010-08-01

    Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. In this investigation, an untranslatable chimeric construct pYP08 containing truncated PRSV coat protein (CP) and PLDMV CP genes coupled with the 3' untranslational region of PLDMV, was generated. Root segments from different portions of adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars 'Tainung No. 2', 'Sunrise', and 'Thailand' were cultured on induction medium for regeneration into somatic embryos. The highest frequency of somatic embryogenesis was from the root-tip segments of adventitious roots developed 2-4 weeks after rooting in perlite medium. After proliferation, embryogenic tissues derived from somatic embryos were wounded in liquid-phase by carborundum and transformed by Agrobacterium carrying pYP08. Similarly, another construct pBG-PLDMVstop containing untranslatable CP gene of PLDMV was also transferred to 'Sunrise' and 'Thailand', the parental cultivars of 'Tainung No. 2'. Among 107 transgenic lines regenerated from 349 root-tip segments, nine lines of Tainung No. 2 carrying YP08 were highly resistant to PRSV and PLDMV, and 9 lines (8 'Sunrise' and 1 'Thailand') carrying PLDMV CP highly resistant to PLDMV, by a mechanism of post-transcriptional gene silencing. The hermaphroditic characteristics of the transgenic lines were confirmed by PCR with sex-linked primers and phenotypes of flower and fruit. Our approach has generated transgenic resistance to both PRSV and PLDMV with commercially desirable characters and can significantly shorten the time-consuming breeding programs for the generation of elite cultivars of papaya hybrids.

  8. Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Directory of Open Access Journals (Sweden)

    Marcela Cristina Correa de Freitas

    2014-06-01

    Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

  9. A novel cell line derived from pleomorphic adenoma expresses MMP2, MMP9, TIMP1, TIMP2, and shows numeric chromosomal anomalies.

    Science.gov (United States)

    Falcão, Aline Semblano Carreira; Kataoka, Maria Sueli da Silva; Ribeiro, Nélson Antonio Bailão; Diniz, José Antonio Picanço; Alves, Sérgio Melo; Ribeiro, André L Ribeiro; de Siqueira, Adriane Sousa; da Silva, Artur Luiz; Ramos, Rommel Thiago Jucá; Freitas, Vanessa M; Jaeger, Ruy G; Pinheiro, João J V

    2014-01-01

    Pleomorphic adenoma is the most common salivary gland neoplasm, and it can be locally invasive, despite its slow growth. This study aimed to establish a novel cell line (AP-1) derived from a human pleomorphic adenoma sample to better understand local invasiveness of this tumor. AP-1 cell line was characterized by cell growth analysis, expression of epithelial and myoepithelial markers by immunofluorescence, electron microscopy, 3D cell culture assays, cytogenetic features and transcriptomic study. Expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) was also analyzed by immunofluorescence and zymography. Furthermore, epithelial and myoepithelial markers, MMPs and TIMPs were studied in the tumor that originated the cell line. AP-1 cells showed neoplastic epithelial and myoepithelial markers, such as cytokeratins, vimentin, S100 protein and smooth-muscle actin. These molecules were also found in vivo, in the tumor that originated the cell line. MMPs and TIMPs were observed in vivo and in AP-1 cells. Growth curve showed that AP-1 exhibited a doubling time of 3.342 days. AP-1 cells grown inside Matrigel recapitulated tumor architecture. Different numerical and structural chromosomal anomalies were visualized in cytogenetic analysis. Transcriptomic analysis addressed expression of 7 target genes (VIM, TIMP2, MMP2, MMP9, TIMP1, ACTA2 e PLAG1). Results were compared to transcriptomic profile of non-neoplastic salivary gland cells (HSG). Only MMP9 was not expressed in both libraries, and VIM was expressed solely in AP-1 library. The major difference regarding gene expression level between AP-1 and HSG samples occurred for MMP2. This gene was 184 times more expressed in AP-1 cells. Our findings suggest that AP-1 cell line could be a useful model for further studies on pleomorphic adenoma biology.

  10. Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.B11

    Directory of Open Access Journals (Sweden)

    Susanne K. Hansen

    2016-05-01

    Full Text Available Spinocerebellar ataxia type 3 (SCA3 is a dominantly inherited neurodegenerative disease caused by an expansion of the CAG-repeat in ATXN3. In this study, induced pluripotent stem cells (iPSCs were generated from SCA3 patient dermal fibroblasts by electroporation with episomal plasmids encoding L-MYC, LIN28, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. Potentially, this iPSC line could be a useful tool for the investigation of SCA3 disease mechanisms.

  11. Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.A11

    Directory of Open Access Journals (Sweden)

    Susanne K. Hansen

    2016-05-01

    Full Text Available Spinocerebellar ataxia type 3 (SCA3 is a dominantly inherited neurodegenerative disease caused by a CAG-repeat expanding mutation in ATXN3. We generated induced pluripotent stem cells (iPSCs from a SCA3 patient by electroporation of dermal fibroblasts with episomal plasmids encoding L-MYC, LIN28, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of genomically integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. This iPSC line could be useful for the investigation of SCA3 disease mechanisms.

  12. A Spirulina maxima-derived peptide inhibits HIV-1 infection in a human T cell line MT4

    Directory of Open Access Journals (Sweden)

    In-Seung Jang

    2016-11-01

    Full Text Available Abstract Human immunodeficiency virus (HIV is the causative agent of acquired immune deficiency syndrome (AIDS. Anti-HIV agents targeting various steps in HIV life cycle have been developed; however, so far, no effective drugs have been found. We show here that a peptide isolated from Spirulina maxima (SM-peptide inhibits HIV-1 infection in a human T cell line MT4. SM-peptide inhibited HIV-1IIIB-induced cell lysis with a half-maximal inhibitory concentration (IC50 of 0.691 mM, while its 50 % cytotoxic concentration (CC50 was greater than 1.457 mM. Furthermore, the SM-peptide inhibited the HIV-1 reverse transcriptase activity and p24 antigen production. This suggests that SM-peptide is a novel candidate peptide, which may be developed as a therapeutic agent for acquired immunodeficiency syndrome patients.

  13. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

    Directory of Open Access Journals (Sweden)

    Clementina Sansone

    Full Text Available Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD, 2-trans,4-trans-octadienal (OD and 2-trans,4-trans-heptadienal (HD on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1 and Fas Associated Death Domain (FADD leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP. The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

  14. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

    Science.gov (United States)

    Sansone, Clementina; Braca, Alessandra; Ercolesi, Elena; Romano, Giovanna; Palumbo, Anna; Casotti, Raffaella; Francone, Maria; Ianora, Adrianna

    2014-01-01

    Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

  15. In Vitro Evaluation of the Antimicrobial Ability and Cytotoxicity on Two Melanoma Cell Lines of a Benzylamide Derivative of Maslinic Acid

    Science.gov (United States)

    Dehelean, Cristina Adriana; Muntean, Delia; Csuk, René

    2016-01-01

    Maslinic acid is a pentacyclic triterpene extracted from olives that has been systematically reported to exert several therapeutic effects, such as antitumoral, antidiabetic, antioxidant, anti-inflammatory, antiparasitic, and antiviral properties. Recently, new derivatives of maslinic acid have been obtained and expanded the spectrum of biological activities and improved the existing ones. The present study was meant to perform the in vitro assessment of the (i) cytotoxic effects of a benzylamide derivative of maslinic acid (“EM2”) (benzyl (2α, 3β) 2,3-diacetoxy-olean-12-en-28-amide) on B164A5 murine melanoma and A375 human malignant melanoma cell lines and the (ii) antimicrobial activity of the compound on several bacterial strains, respectively. We obtained a dose-dependent cytotoxic effect of EM2 that was particularly relevant to the murine cell line. As on the antibacterial activity, EM2 was tested on 10 bacterial strains Bacillus cereus, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, Escherichia coli, Yersinia enterocolitica, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa and one fungus Candida albicans. A significant antimicrobial effect was recorded for Streptococcus pyogenes and Staphylococcus aureus. PMID:28050335

  16. In Vitro Evaluation of the Antimicrobial Ability and Cytotoxicity on Two Melanoma Cell Lines of a Benzylamide Derivative of Maslinic Acid

    Directory of Open Access Journals (Sweden)

    Ioana Zinuca Pavel

    2016-01-01

    Full Text Available Maslinic acid is a pentacyclic triterpene extracted from olives that has been systematically reported to exert several therapeutic effects, such as antitumoral, antidiabetic, antioxidant, anti-inflammatory, antiparasitic, and antiviral properties. Recently, new derivatives of maslinic acid have been obtained and expanded the spectrum of biological activities and improved the existing ones. The present study was meant to perform the in vitro assessment of the (i cytotoxic effects of a benzylamide derivative of maslinic acid (“EM2” (benzyl (2α, 3β 2,3-diacetoxy-olean-12-en-28-amide on B164A5 murine melanoma and A375 human malignant melanoma cell lines and the (ii antimicrobial activity of the compound on several bacterial strains, respectively. We obtained a dose-dependent cytotoxic effect of EM2 that was particularly relevant to the murine cell line. As on the antibacterial activity, EM2 was tested on 10 bacterial strains Bacillus cereus, Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Enterococcus faecalis, Escherichia coli, Yersinia enterocolitica, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa and one fungus Candida albicans. A significant antimicrobial effect was recorded for Streptococcus pyogenes and Staphylococcus aureus.

  17. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    Science.gov (United States)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  18. A novel approach to evaluate the pharmacokinetic biocomparability of a monoclonal antibody derived from two different cell lines using simultaneous crossover design.

    Science.gov (United States)

    Han, Chao; McIntosh, Thomas S; Geist, Brian J; Jiao, Trina; Puchalski, Thomas A; Goldberg, Kenneth M; Yang, Tong-Yuan; Pendley, Charles E; Zhou, Honghui; Davis, Hugh M

    2014-01-01

    A parallel study design with a large number of subjects has been a typical path for pharmacokinetic (PK) biocomparability assessment of biotherapeutics with long half-lives and immunogenic propensity, for example, monoclonal antibodies (mAb). A recently published innovative bioanalytical method that can quantify mAb produced from two different cell lines in the same sample opened an avenue to exploring a simultaneous crossover study design for PK biocomparability assessment of biotherapeutics. Siltuximab, a chimeric IgG1 mAb-targeting interleukin-6, was studied as an example. The pharmacokinetic biocomparability of siltuximab derived from mouse myeloma (Sp2/0) cells and Chinese hamster ovary cells was previously assessed and demonstrated in a clinical PK biocomparability study that enrolled more than 140 healthy subjects using a parallel trial design. The biocomparability was successfully shown in six cynomolgus monkeys in a preclinical proof-of-concept study using the new crossover study design supported by the analytical method. The impact of antidrug antibodies on the assessment of biocomparability was minimal. This novel approach opened up a new arena for the evaluation of PK biocomparability of biotherapeutics with unique molecular signatures such as a mAb derived from different cell lines.

  19. Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

    Directory of Open Access Journals (Sweden)

    Ponti Donatella

    2011-03-01

    Full Text Available Abstract Background Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1 predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics. Materials and methods Cytotoxicity assays were performed on cells derived from fresh tumor explants of 18 human cases of malignant glioma. In addition to EGR-1, tumor cultures were investigated for genetic alterations and the expression of cancer regulating factors, related to the p53 pathway. Results We found that sensitivity to cisplatin correlates significantly with levels of EGR-1 expression in tumors with wild-type p53/INK4a/p16 status. Conclusion Increased knowledge of the mechanisms regulating EGR-1 expression in wild-type p53/INK4a/p16 cases of glioma may help in the design of new chemotherapeutic strategies for these tumors.

  20. Determination of Aniline and Its Derivatives in Environmental Water by Capillary Electrophoresis with On-Line Concentration

    Directory of Open Access Journals (Sweden)

    Jianzhi Sun

    2012-06-01

    Full Text Available This paper describes a simple, sensitive and environmentally benign method for the direct determination of aniline and its derivatives in environmental water samples by capillary zone electrophoresis (CZE with field-enhanced sample injection. The parameters that influenced the enhancement and separation efficiencies were investigated. Surprisingly, under the optimized conditions, two linear ranges for the calibration plot, 1–50 ng/mL and 50–1000 ng/mL (R > 0.998, were obtained. The detection limit was in the range of 0.29–0.43 ng/mL. To eliminate the effect of the real sample matrix on the stacking efficiency, the standard addition method was applied to the analysis of water samples from local rivers.

  1. Flavokawain derivative FLS induced G2/M arrest and apoptosis on breast cancer MCF-7 cell line.

    Science.gov (United States)

    Ali, Norlaily Mohd; Akhtar, M Nadeem; Ky, Huynh; Lim, Kian Lam; Abu, Nadiah; Zareen, Seema; Ho, Wan Yong; Alan-Ong, Han Kiat; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Ismail, Jamil Bin; Yeap, Swee Keong; Kamarul, Tunku

    2016-01-01

    Known as naturally occurring biologically active compounds, flavokawain A and B are the leading chalcones that possess anticancer properties. Another flavokawain derivative, (E)-1-(2'-Hydroxy-4',6'-dimethoxyphenyl)-3-(4-methylthio)phenyl)prop-2-ene-1-one (FLS) was characterized with (1)H-nuclear magnetic resonance, electron-impact mas spectrometry, infrared spectroscopy, and ultraviolet ((1)H NMR, EI-MS, IR, and UV) spectroscopic techniques. FLS cytotoxic efficacy against human cancer cells (MCF-7, MDA-MB-231, and MCF-10A) resulted in the reduction of IC50 values in a time- and dose-dependent mode with high specificity on MCF-7 (IC50 of 36 μM at 48 hours) against normal breast cell MCF-10A (no IC50 detected up to 180 μM at 72 hours). Light, scanning electron, and fluorescent microscopic analysis of MCF-7 cells treated with 36 μM of FLS displayed cell shrinkage, apoptotic body, and DNA fragmentation. Additionally, induction of G2/M cell arrest within 24 hours and apoptosis at subsequent time points was discovered via flow cytometry analysis. The roles of PLK-1, Wee-1, and phosphorylation of CDC-2 in G2/M arrest and proapoptotic factors (Bax, caspase 9, and p53) in promotion of apoptosis of FLS against MCF-7 cells were discovered using fluorometric, quantitative real-time polymerase chain reaction, and Western blot analysis. Interestingly, the presence of SCH3 (thiomethyl group) on ring B structure contributed to the selective cytotoxicity against MCF-7 cells compared to other chalcones, flavokawain A and B. Overall, our data suggest potential therapeutic value for flavokawain derivative FLS to be further developed as a new anticancer drug.

  2. Derivation of feline vaccine-associated fibrosarcoma cell line and its growth on chick embryo chorioallantoic membrane - a new in vivo model for veterinary oncological studies.

    Science.gov (United States)

    Zabielska, K; Lechowski, R; Król, M; Pawłowski, K M; Motyl, T; Dolka, I; Zbikowski, A

    2012-12-01

    Feline vaccine associated fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated fibrosarcoma cell line and its growth on CAM is described. The cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.

  3. Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yoon Hee [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Gupta, Mukesh Kumar, E-mail: goops@konkuk.ac.kr [Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Oh, Shin Hye [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Uhm, Sang Jun [Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Lee, Hoon Taek, E-mail: htl3675@konkuk.ac.kr [Department of Bioscience and Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of); Department of Animal Biotechnology, Bio-Organ Research Center/Animal Resources Research Center, Konkuk University, Hwayang-dong, Gwangjin-Gu, Seoul 143 701 (Korea, Republic of)

    2010-03-10

    This study evaluated the essentiality of glial cell line-derived neurotrophic factor (GDNF) for in vitro culture of established mouse multipotent adult germline stem (maGS) cell lines by culturing them in the presence of GDNF, leukemia inhibitory factor (LIF) or both. We show that, in the absence of LIF, GDNF slows the proliferation of maGS cells and result in smaller sized colonies without any change in distribution of cells to different cell-cycle stages, expression of pluripotency genes and in vitro differentiation potential. Furthermore, in the absence of LIF, GDNF increased the expression of male germ-line genes and repopulated the empty seminiferous tubule of W/W{sup v} mutant mouse without the formation of teratoma. GDNF also altered the genomic imprinting of Igf2, Peg1, and H19 genes but had no effect on DNA methylation of Oct4, Nanog and Stra8 genes. However, these effects of GDNF were masked in the presence of LIF. GDNF also did not interfere with the multipotency of maGS cells if they are cultured in the presence of LIF. In conclusion, our results suggest that, in the absence of LIF, GDNF alters the growth characteristics of maGS cells and partially impart them some of the germline stem (GS) cell-like characteristics.

  4. Synthesis and biological evaluation of some substituted-2-N-(5-chloro-2-methoxy-4-methylphenylsulphonyl) glutamic acid derivatives against prostate cancer cell line PC3.

    Science.gov (United States)

    Hassan, Ghaneya Sayed; Abdel Rahman, Doaa Ezzat

    2013-01-01

    New series of substituted glutamine 5a-l and glutamic acid diamides, diureide and dihydrazide 7a-e were synthesized from parent glutamic acid compound 3 and evaluated for their cytotoxic activity against tumor cell line PC3 (prostate cancer cell line). Most of the tested compounds exploited potent growth inhibitory activity with IC(50) values ranging 0.034-3.97 µM. Particularly, compounds 5a, 3, 5j, 5b, 7c, 7e, 5l, and 5k exhibited superior potency (IC(50)=0.034, 0.04, 0.05, 0.074, 0.25, 0.4, 0.49, 0.522 µM, respectively) to the reference drug Doxorubicin (IC(50)=0.63 µM), while compound 7b showed IC(50), 0.71 µM, comparable to that of Doxorubicin. In summary, the newly synthesized compounds provided promising new lead for the future design and development of glutamine and glutamic acid derivatives as novel antitumor agents. The quantitative structure-activity relationship (QSAR) study was applied to find a mathematical correlation between the structures of compounds and their activity against PC3 cell line expressed as IC(50) values.

  5. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

    Science.gov (United States)

    Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

    2017-03-15

    Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Long-term mean and seasonal variation of altimetry-derived Oyashio transport across the A-line off the southeastern coast of Hokkaido, Japan

    Science.gov (United States)

    Kuroda, Hiroshi; Wagawa, Taku; Kakehi, Shigeho; Shimizu, Yugo; Kusaka, Akira; Okunishi, Takeshi; Hasegawa, Daisuke; Ito, Shin-ichi

    2017-03-01

    To understand long-term mean and seasonal variations of the Oyashio transport off the southeastern coast of Hokkaido, time series of estimated transports were generated by combining satellite altimetry and CTD data from the period 1993-2014 along a monitoring transect referred to as the ;A-line;. Linear regressions between CTD-derived geostrophic transport and altimetry-derived sea level anomaly differences were calculated by minimizing a cost function newly proposed in this study. The horizontal structure of 21-year mean transport was characterized by a southwestward flow on the continental slope (SW-1) associated with the First Oyashio Intrusion, a northeastward flow (NE-2) likely trapped against the Kuril-Kamchatka trench, a southwestward flow (SW-3) related to the Second Oyashio Intrusion, and a very stable northeastward flow (NE-4) corresponding to a combination of the Oyashio Return Flow and quasi-stationary Jet. Analysis of long-term mean monthly transports revealed four alternating flows that were present throughout all seasons of the year. The boundaries between adjacent pairs of the four flows on the A-line did not change on a seasonal timescale as much as the southernmost latitude of the First Oyashio Intrusion. At a 0-month (1-month) time lag, the sum of the SW-1, NE-2, and SW-3 (SW-1 and NE-2) transports, which corresponds to net Oyashio transport (partial net Oyashio transport related to the southernmost latitude of the First Oyashio Intrusion), was correlated with Sverdrup transport more robustly than was each of the four alternating flows. This result suggests that as the Oyashio crossed the A-line, it responded barotropically to basin-scale wind stress by adjusting transports composed of both southwestward and northeastward flows.

  7. Glial cell line-derived neurotrophic factor attenuates behavioural deficits and regulates nigrostriatal dopaminergic and peptidergic markers in 6-hydroxydopamine-lesioned adult rats: comparison of intraventricular and intranigral delivery.

    Science.gov (United States)

    Lapchak, P A; Miller, P J; Collins, F; Jiao, S

    1997-05-01

    The effects of intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor were tested on low dose (0.05 mg/kg) apomorphine-induced rotations and tyrosine hydroxylase activity in the substantia nigra and striatum of stable 6-hydroxydopamine-lesioned rats. In addition, we determined if 6-hydroxydopamine lesions in the absence or presence of treatment affected neuropeptide (substance P, met-enkephalin, dynorphin) content in the striatum. Glial cell line-derived neurotrophic factor, when administered intranigrally, prevented apomorphine-induced rotational behaviour for 11 weeks following a single injection. In comparison, intraventricularly-administered glial cell line-derived neurotrophic factor produced a transient reduction in rotational behaviour that lasted for two to three weeks following a single injection. We also show that rotational behaviour is reduced following each subsequent intraventricular injection of glial cell line-derived neurotrophic factor given every six weeks, a time-point when baseline rotation deficits were re-established. Intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor signifi