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Sample records for line c4-2 derived

  1. Anomalous Dispersion in Gases Derived from the Optical Depth. Theoretical Treatment: Line by Line Calculations

    DTIC Science & Technology

    1991-06-28

    AD-A238 853 ANOMALOUS DISPERSION IN GASES DERIVED FROM THE OPTICAL DEPTH. THEORETICAL TREATMENT; LINE BY LINE CALCULATIONS BY EGIL BINGEN . BJ0RNAR...06054 917 1 04 ANOMALOUS DISPERSION IN GASES DERIVED FROM THE OPTICAL DEPTH. THEORETICAL TREATMENT; LINE BY LINE CALCULATIONS by - EGII, BINGEN . BJORNAR... BINGEN Egil, YSTAD Bjornar 61 DISTRIBUTION STATEMENT Approved for pub’ic release. Distribution unlimited (Offentlig tilgjengelig) 7) INDEXING TERMS IN

  2. A novel subfamily of LINE-derived elements in mice.

    PubMed

    Flood, W D; Rogozin, I B; Ruvinsky, A

    1998-11-01

    Hybrid sequences have been described previously that consist of a 5' region homologous to ORF2 of LINEs and a 3' end that shares homology with a sequence located in the first intron of Cepsilon immunoglobulin. The present investigation has revealed 14 new sequences from seven murine species, that show high homology to those observed earlier. Database search has found several new homologous hybrid sequences including one located in the mouse T-cell receptor (Tcra) locus. Several interesting features of this sequence include identical 15-bp flanking short direct repeats as well as poly-A signal and A-rich sequence at the 3' end. We have classified this set of sequences as LINE-derived elements (LDEs), which constitute a newly observed subfamily. Comparative analysis of these sequences suggests that a single recombination event was responsible for the production of an LDE progenitor. The phylogenetic tree shows a number of elements that pre-existed in the common ancestor of murine species and displays different evolutionary rates. The time of LDE origin is estimated at approximately 10-15 MYA.

  3. LINE DERIVED INFRARED EXTINCTION TOWARD THE GALACTIC CENTER

    SciTech Connect

    Fritz, T. K.; Gillessen, S.; Dodds-Eden, K.; Lutz, D.; Genzel, R.; Raab, W.; Ott, T.; Pfuhl, O.; Eisenhauer, F.; Yusef-Zadeh, F.

    2011-08-20

    We derive the extinction curve toward the Galactic center (GC) from 1 to 19 {mu}m. We use hydrogen emission lines of the minispiral observed by ISO-SWS and SINFONI. The extinction-free flux reference is the 2 cm continuum emission observed by the Very Large Array. Toward the inner 14'' x 20'', we find an extinction of A{sub 2.166{mu}m} = 2.62 {+-} 0.11, with a power-law slope of {alpha} = -2.11 {+-} 0.06 shortward of 2.8 {mu}m, consistent with the average near-infrared slope from the recent literature. At longer wavelengths, however, we find that the extinction is grayer than shortward of 2.8 {mu}m. We find that it is not possible to fit the observed extinction curve with a dust model consisting of pure carbonaceous and silicate grains only, and the addition of composite particles, including ices, is needed to explain the observations. Combining a distance-dependent extinction with our distance-independent extinction, we derive the distance to the GC to be R{sub 0} = 7.94 {+-} 0.65 kpc. Toward Sgr A* (r < 0.''5), we obtain A{sub H} = 4.21 {+-} 0.10, A{sub Ks} = 2.42 {+-} 0.10, and A{sub L'} = 1.09 {+-} 0.13.

  4. Brain-derived and glial cell line-derived neurotrophic factor fusion protein immobilization to laminin

    PubMed Central

    Wang, Baoxin; Yuan, Junjie; Xu, Jiafeng; Chen, Xinwei; Ying, Xinjiang; Dong, Pin

    2017-01-01

    Damage to the recurrent laryngeal nerve often causes hoarseness, dyspnea, dysphagia, and sometimes asphyxia due to vocal cord paralysis which result in a reduction of quality of life. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) play critical roles in peripheral nerve regeneration. However, methods for efficiently delivering these molecules are lacking, which limits their use in clinical applications. The present study reports an effective strategy for targeting BDNF and GDNF to laminin by fusing the N-terminal domains of these molecules with agrin (NtA). More specifically, laminin-binding efficacy was assessed and sustained release assays of the delivery of BDNF or GDNF fused with NtA (LBD-BDNF or LBD-GDNF) to laminin were conducted in vitro. In addition, the bioactivity of LBD-BDNF and LBD-GDNF on laminin in vitro was investigated. LBD-BDNF and LBD-GDNF were each able to specifically bind to laminin and maintain their activity in vitro. Moreover, neurotrophic factors with NtA retained higher concentrations and bioactivity levels compared with those without NtA. The ratio of LBD-BDNF and LBD-GDNF that produced optimal effects was 4:6. BDNF and GDNF fused with NtA were effective in specifically binding to laminin. As laminin is a major component of the extracellular matrix, LBD-BDNF and LBD-GDNF may prove useful in the repair of peripheral nerve injuries. PMID:28123487

  5. Tangent Lines without Derivatives for Quadratic and Cubic Equations

    ERIC Educational Resources Information Center

    Carroll, William J.

    2009-01-01

    In the quadratic equation, y = ax[superscript 2] + bx + c, the equation y = bx + c is identified as the equation of the line tangent to the parabola at its y-intercept. This is extended to give a convenient method of graphing tangent lines at any point on the graph of a quadratic or a cubic equation. (Contains 5 figures.)

  6. Efficient derivation of extraembryonic endoderm stem cell lines from mouse postimplantation embryos

    PubMed Central

    Lin, Jiangwei; Khan, Mona; Zapiec, Bolek; Mombaerts, Peter

    2016-01-01

    Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5. PMID:27991575

  7. Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats.

    PubMed

    Men, Hongsheng; Bauer, Beth A; Bryda, Elizabeth C

    2012-09-20

    Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line-derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  9. Impaired dental cytodifferentiation in glial cell-line derived growth factor (GDNF) deficient mice.

    PubMed

    de Vicente, J C; Cabo, R; Ciriaco, E; Laurà, R; Naves, F J; Silos-Santiago, I; Vega, J A

    2002-01-01

    Glial cell line-derived neurotrophic factor promotes the survival of multiple neuron types in the central and peripheral nervous system. Moreover, it plays a key role in the development of the enteric nervous system and in the kidney organogenesis. Glial cell line-derived neurotrophic factor and their receptors are expressed in the developing tooth as well as in the trigeminal ganglion. However, the precise role of this growth factor in tooth morphogenesis and cell differentiation, or in the development of trigeminal ganglion cells, is still elusive. Using structural and ultrastructural techniques we analyzed in detail the first molar tooth germ of glial cell line-derived neurotrophic factor deficient mice as well as the neuronal density in trigeminal ganglion. The length and width of first molar tooth germ in knockout deficient animals showed no differences in the knockout animals in comparison with age-matched heterozygous or wild-type littermates. Nevertheless, in mice lacking glial cell line-derived neurotrophic factor, both ameloblasts and odontoblasts failed to fully develop and differentiate, and the enamel matrix and predentin layers were absent. On the other hand, the number of trigeminal sensory neurons and the structure of the nerves supplying first molar tooth germ were largely normal. Present results suggest a new non-neuronal role for glial cell line-derived neurotrophic factor in tooth development. Glial cell line-derived neurotrophic factor seems not to be involved in tooth initiation and morphogenesis, whereas it seems essential for cytodifferentiation. Conversely, neither development of trigeminal neuron nor nerve fibers supplying teeth are directly dependent on glial cell line-derived neutrophic factor.

  10. Successful derivation of xeno-free mesenchymal stem cell lines from endometrium of infertile women.

    PubMed

    Phermthai, Tatsanee; Tungprasertpol, Kittima; Julavijitphong, Suphakde; Pokathikorn, Puttachart; Thongbopit, Sasiprapa; Wichitwiengrat, Suparat

    2016-12-01

    Transplantation of mesenchymal stem cells (MSC) can effectively repair endometrial deficiencies, including infertile patients with a problem of inadequate endometrium thickness. Although, MSC derived from different organ sources have a similarity of MSC specific characteristics, endometrial stem cells (EMSC) are temporally regulated throughout the menstrual cycle in a micro-environmental niche found only in endometrial tissue. Given the micro-environment niche, developing treatments for endometrial disorders with EMSC should be a top priority. To provide EMSC that afford safety for therapeutic usage, we have established a completely xeno-free EMSC line derivation protocol using human allogenic umbilical cord serum instead of animal derived reagents, and proved that it is feasible to generate xeno-free EMSC lines from infertile patient donors using these conditions. Our results demonstrate the successful derivation of xeno-free EMSC lines from 10 out of 10 infertile patients. The resultant xeno-free EMSC lines showed typical MSC morphology, phenotypic markers, differentiation capacity, telomere length and normal karyotypes. They showed superior proliferation capability, but lower expression of proto-oncogenes, to the lines generated under standard (animal derived reagents) culture. Biosafety of xeno-free EMSC lines also displayed in retention of immunosuppressive ability, epigenetic stability by imprinted genes expression, proto-oncogenes expression and no mutation of specific codon on p53 tumor suppressor gene. Taken together, these data indicate that our cells may be safe for clinical use. In conclusion, we have succeeded in establishing completely xeno-free EMSC lines and demonstrate for the first time that autogenic and xeno-free EMSC lines can be generated from infertile women.

  11. Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

    PubMed Central

    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. PMID:24797371

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    CORRÊA, NATÁSSIA C.R.; KUASNE, HELLEN; FARIA, JERUSA A.Q.A.; SEIXAS, CIÇA C.S.; SANTOS, IRIA G.D.; ABREU, FRANCINE B.; NONOGAKI, SUELY; ROCHA, RAFAEL M.; SILVA, GERLUZA APARECIDA BORGES; GOBBI, HELENICE; ROGATTO, SILVIA R.; GOES, ALFREDO M.; GOMES, DAWIDSON 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 in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. 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 to be potentially used for comparative research. PMID:23404580

  15. Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos.

    PubMed

    Li, Chunliang; Yang, Ying; Lu, Xiaowei; Sun, Yijuan; Gu, Junjie; Feng, Yun; Jin, Ying

    2010-04-01

    Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research as well as a potential cell resource for therapy. However, each hES cell line demonstrates different identity. It is desirable to obtain more fully characterized hES cell lines with newly developed technologies associated with hES cell culture. Here, we report our experience of efficient derivation of three new Chinese hES cell lines (SHhES2, SHhES3, and SHhES4) from in vitro fertilization discarded embryos donated by women with polycystic ovary syndrome. These cell lines were derived under conditions minimizing exposure to animal components and maintained at an undifferentiated state for long-term culture. They retained a normal karyotype and expressed ALP, OCT4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. RT-PCR analysis also revealed high expression levels of pluripotency markers such as OCT4, LEFTY A, SOX2, TDGF-1, THY1, FGF4, NANOG, and REX1. When suspended in low-attachment culture dishes, embryoid bodies formed and were comprised of various differentiated cell types from all three embryonic germ layers. However, well-shaped teratomas were only harvested from line SHhES2, not from SHhES3 and SHhES4, indicating that the differentiation ability in vivo differs among the three cell lines. Collectively, the three new hES cell lines were established and fully characterized. The effort paves the way toward generating hES cell lines without contamination by animal components. All of these cell lines are available by contact Ying Jin at yjin@sibs.ac.cn.

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

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    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.

  17. Derivation of Huntington Disease affected Genea091 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Schaft, Julia; McKernan, Robert; Hu, Jesselyn; Schmidt, Uli

    2016-03-01

    The Genea091 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 40 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, 92% of cells expressed Nanog, 97% Oct4, 79% Tra1-60 and 98% SSEA4 and gave a Pluritest pluripotency score of 38.36, Novelty of 1.35. The cell line was negative for Mycoplasma and visible contamination.

  18. Derivation of Huntington Disease affected Genea089 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; McKernan, Robert; Hu, Jesselyn; Schmidt, Uli

    2016-03-01

    The Genea089 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 41 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, 91% of cells expressed Nanog, 95% Oct4, 90% Tra1-60 and 100% SSEA4 and gave a PluriTest Pluripotency score of 39.28, Novelty of 1.2. The cell line was negative for Mycoplasma and visible contamination.

  19. Derivation of Huntington disease affected Genea020 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Peura, Teija; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea020 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 48 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, 89% of cells expressed Nanog, 95% Oct4, 29% Tra1-60 and 99% SSEA4, gave a Pluritest pluripotency score of 27.51, novelty of 1.43 and demonstrated alkaline phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  20. Derivation of Huntington Disease affected Genea018 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Main, Heather; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea018 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 46 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, 75% of cells expressed Nanog, 91% Oct4, 73% Tra1-60 and 96% SSEA4, gave a Pluritest pluripotency score of 31.12, Novelty of 1.45, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    PubMed Central

    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

  3. In vivo behavior of murine epidermal cell lines derived from initiated and noninitiated skin.

    PubMed

    Conti, C J; Fries, J W; Viaje, A; Miller, D R; Morris, R; Slaga, T J

    1988-01-15

    The in vivo behavior of cell cultures derived from normal and carcinogen-treated mouse epidermis was studied by implanting the cultures in a s.c. vascularized bed protected by a silicone chamber. Cells derived from normal adult mouse epidermis as well as cells derived from tumor-promoter-treated skin were unable to grow in these systems. Conversely, cell lines derived from skin initiated with single doses of N-methyl-N'-nitro-N-nitrosoguanidine or 9,10-dimethyl-1,2-benzanthracene proliferated in these chambers, reforming an epithelial structure. The type of structure in the chambers varied, ranging from formation of almost normal epithelia to atypical invasive behavior. The variable in vivo behavior among the different cell lines may be attributed to the initiation agent, the number of passages of the cultures, random genetic events, the strain of mouse, or a combination of these factors. Most of the cell types used in this study and all the cell lines that were able to grow in these chambers were selected for resistance to Ca-induced terminal differentiation. However, resistance to terminal differentiation according to the Ca2+ switch does not always correlate with the ability to grow in the chambers, since cell lines derived from spontaneous foci of resistance failed to grow in this system. These studies showed some of the possibilities of the SC silicone chambers to study the histogenic potential of cell lines derived from carcinogen-treated epidermis. This system also appears suitable to study the complex relationship between epidermal cells and specialized (dermal) stroma.

  4. Genetic mapping with an inbred line-derived F2 population in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato (Solanum tuberosum L.) is an important global food crop, for which tetrasomic inheritance and self-incompatibility have limited both genetic discovery and breeding gains. We report here on the creation of the first diploid inbred line-derived F2 population in potato, and demonstrate its utili...

  5. Characterization of the Recombinant Inbred Line Population Derived from the Cross of Nipponbare/9311

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As a part of the project entitled “Understanding the rice epigenome: From genes to genomes” funded by the National Science Foundation, a mapping population of 480 F6-8 recombinant inbred lines (RILs) derived from a cross of Nipponbare with 9311 (Nip/9311) was developed. Phenotyping important agronom...

  6. The lateral line confers evolutionarily derived sleep loss in the Mexican cavefish.

    PubMed

    Jaggard, James; Robinson, Beatriz G; Stahl, Bethany A; Oh, Ian; Masek, Pavel; Yoshizawa, Masato; Keene, Alex C

    2017-01-15

    Sleep is an essential behavior exhibited by nearly all animals, and disruption of this process is associated with an array of physiological and behavioral deficits. Sleep is defined by changes in sensory gating that reduce sensory input to the brain, but little is known about the neural basis for interactions between sleep and sensory processing. Blind Mexican cavefish comprise an extant surface dwelling form and 29 cave morphs that have independently evolved increased numbers of mechanoreceptive lateral line neuromasts and convergent evolution of sleep loss. Ablation of the lateral line enhanced sleep in the Pachón cavefish population, suggesting that heightened sensory input underlies evolutionarily derived sleep loss. Targeted lateral line ablation and behavioral analysis localized the wake-promoting neuromasts in Pachón cavefish to superficial neuromasts of the trunk and cranial regions. Strikingly, lateral line ablation did not affect sleep in four other cavefish populations, suggesting that distinct neural mechanisms regulate the evolution of sleep loss in independently derived cavefish populations. Cavefish are subject to seasonal changes in food availability, raising the possibility that sensory modulation of sleep is influenced by metabolic state. We found that starvation promotes sleep in Pachón cavefish, and is not enhanced by lateral line ablation, suggesting that functional interactions occur between sensory and metabolic regulation of sleep. Taken together, these findings support a model where sensory processing contributes to evolutionarily derived changes in sleep that are modulated in accordance with food availability.

  7. Derivation of a continuum model and the energy law for moving contact lines with insoluble surfactants

    SciTech Connect

    Zhang, Zhen Xu, Shixin; Ren, Weiqing

    2014-06-15

    A continuous model is derived for the dynamics of two immiscible fluids with moving contact lines and insoluble surfactants based on thermodynamic principles. The continuum model consists of the Navier-Stokes equations for the dynamics of the two fluids and a convection-diffusion equation for the evolution of the surfactant on the fluid interface. The interface condition, the boundary condition for the slip velocity, and the condition for the dynamic contact angle are derived from the consideration of energy dissipations. Different types of energy dissipations, including the viscous dissipation, the dissipations on the solid wall and at the contact line, as well as the dissipation due to the diffusion of surfactant, are identified from the analysis. A finite element method is developed for the continuum model. Numerical experiments are performed to demonstrate the influence of surfactant on the contact line dynamics. The different types of energy dissipations are compared numerically.

  8. Expression of P2Y receptors in cell lines derived from the human lung.

    PubMed

    Communi, D; Paindavoine, P; Place, G A; Parmentier, M; Boeynaems, J M

    1999-05-01

    1. Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo- and 16HBE14o- epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU-3 serous cells, the 6CFSMEo- submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC-1. These experiments revealed that P2Y2 and P2Y6 mRNA are co-expressed in the IHAEo-, 16HBE14o- and A549 epithelial cell lines. The CFPAC-1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors. 2. We have then performed RT-PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo-, 16HBE14o-, 6CFSMEo-, HASMSC1 and CFPAC-1. P2Y1 mRNA was only detected in the CALU-3 cell line. 3. Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo- and the 16HBE14o- airway epithelial cell lines which co-express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo- submucosal cells expressed a UTP-specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

  9. [The construction of urine-derived cell lines from patients with spinal muscular atrophy].

    PubMed

    Wanjin, Chen; Qijie, Zhang; Jin, He; Xiang, Lin; Ning, Wang

    2014-11-01

    Spinal muscular atrophy (SMA) is a common neurodegenerative disease in childhood and infancy, clinically characterized by progressive and symmetric muscular weakness and atrophy. Few effective therapies are available now, and SMA is one of the most common genetic causes of infantile mortality. SMA patient-derived cells are beneficial in basic research on this disease, but the most common model cell, fibroblasts can only be obtained through invasive procedures such as muscle or skin biopsy, which are unwelcome to patients and their families. In this study, fresh urine from SMA patients and healthy controls was collected and centrifuged, and the urine sediment was cultured in vitro. The growth characteristics of urine-derived cells were observed, and the survival of motor neuron (SMN) gene, and the amount and localization of SMN protein in different urine cell lines were investigated. In total, 25 urine cell lines from 11 SMA patients and 14 healthy controls were established. These urine-derived cells expand robustly in vitro with stable cell morphological characteristics. The urine cell lines derived from patients carry the SMN1 gene defect and express a low level of SMN protein, while the intracellular localization of SMN protein is normal. Urine-derived cell culture technology is simple, non-invasive and highly reproducible, a way of obtaining and storing rare cell samples from SMA patients with which to study the pathogenesis of SMA.

  10. An investigation of the RWPE prostate derived family of cell lines using FTIR spectroscopy.

    PubMed

    Baker, M J; Clarke, C; Démoulin, D; Nicholson, J M; Lyng, F M; Byrne, H J; Hart, C A; Brown, M D; Clarke, N W; Gardner, P

    2010-05-01

    Interest in developing robust, quicker and easier diagnostic tests for cancer has lead to an increased use of Fourier transform infrared (FTIR) spectroscopy to meet that need. In this study we present the use of different experimental modes of infrared spectroscopy to investigate the RWPE human prostate epithelial cell line family which are derived from the same source but differ in their mode of transformation and their mode of invasive phenotype. Importantly, analysis of the infrared spectra obtained using different experimental modes of infrared spectroscopy produces similar results. The RWPE family of cell lines can be separated into groups based upon the method of cell transformation rather than the resulting invasiveness/aggressiveness of the cell line. The study also demonstrates the possibility of using a genetic algorithm as a possible standardised pre-processing step and raises the important question of the usefulness of cell lines to create a biochemical model of prostate cancer progression.

  11. Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines

    PubMed Central

    2013-01-01

    Background WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the “canonical” WNT/β-catenin signaling pathway, and the “non-canonical” pathways including WNT/Ca2+ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood. The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60. Methods We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR. Results WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB

  12. Identification of peanut agglutinin binding glycoproteins restricted to Hodgkin's disease-derived cell lines.

    PubMed

    Flavell, D J; Jones, D B; Wright, D H

    1989-01-01

    Peanut agglutinin (PNA) binding glycoproteins from four Hodgkin's disease (HD)-derived cell lines and a variety of cell lines/peripheral blood cells representative of the lymphoid and myeloid lineages were identified by probing nitrocellulose membranes of SDS-PAGE separated NP40 solubilized cellular glycoproteins with [125I]-labelled PNA. The two Hodgkin's cell lines Ho and L428 demonstrated the most heterogeneous glycoprotein profiles each expressing 15 PNA binding glycoproteins, respectively. The two remaining Hodgkin's lines Co and L591 expressed only four glycoproteins each and these were all also commonly expressed by Ho and L428. Comparative analysis with all other cell types studied revealed the expression of five glycoproteins restricted to Ho (gp42, gp40, gp38, gp24 and gp22) and six restricted to L428 (gp180, gp75, gp40, gp38, gp24 and gp22). Four of these, gp40, gp38, gp24 and gp22 were commonly expressed by both Ho and L428. Of cell lines of myeloid lineage studied only the erythroleukemia cell line K562 expressed detectable glycoproteins also expressed by some of the Hodgkin's cell lines (gp110, gp96, gp50 and gp45). Only one glycoprotein, gp20 expressed by Ho was also commonly expressed by normal peripheral blood granulocytes. This limited study has thus succeeded in demonstrating for the range of cell types studied, that some glycoproteins with terminal D-galactose beta (1----3) N-acetyl galactosamine oligosaccharide sequences are apparently restricted to two of the HD cell lines. Moreover, the heterogeneous glycoprotein profiles obtained for the HD cell lines Ho and L428 suggests that galactosylation processes in these two cell lines is aberrant.

  13. Tumor-Derived Cell Lines as Molecular Models of Cancer Pharmacogenomics.

    PubMed

    Goodspeed, Andrew; Heiser, Laura M; Gray, Joe W; Costello, James C

    2016-01-01

    Compared with normal cells, tumor cells have undergone an array of genetic and epigenetic alterations. Often, these changes underlie cancer development, progression, and drug resistance, so the utility of model systems rests on their ability to recapitulate the genomic aberrations observed in primary tumors. Tumor-derived cell lines have long been used to study the underlying biologic processes in cancer, as well as screening platforms for discovering and evaluating the efficacy of anticancer therapeutics. Multiple -omic measurements across more than a thousand cancer cell lines have been produced following advances in high-throughput technologies and multigroup collaborative projects. These data complement the large, international cancer genomic sequencing efforts to characterize patient tumors, such as The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC). Given the scope and scale of data that have been generated, researchers are now in a position to evaluate the similarities and differences that exist in genomic features between cell lines and patient samples. As pharmacogenomics models, cell lines offer the advantages of being easily grown, relatively inexpensive, and amenable to high-throughput testing of therapeutic agents. Data generated from cell lines can then be used to link cellular drug response to genomic features, where the ultimate goal is to build predictive signatures of patient outcome. This review highlights the recent work that has compared -omic profiles of cell lines with primary tumors, and discusses the advantages and disadvantages of cancer cell lines as pharmacogenomic models of anticancer therapies.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27009863

  16. Comparison of the sensitivity of three lung derived cell lines to metals from combustion derived particulate matter.

    PubMed

    Riley, Mark R; Boesewetter, Dianne E; Turner, Rachael A; Kim, Aana M; Collier, Jayne M; Hamilton, Amy

    2005-04-01

    While the effects of inhalation of combustion-derived particulate matter have received extensive study, there remains no reliable means to rapidly quantify inhalation toxicity outside of a laboratory setting. Cell-based biosensors provide a potential solution, but few comparisons have been made of the sensitivity of various cell lines to the wide range of inhalation health hazards that are likely to be encountered. This work compares the response of three immortalized lung cell lines (A549 human epithelia, RLE-6TN rat type II epithelia, and NR8383 rat alveolar macrophages) to metals commonly present in combustion-derived particulate matter. Quantifications of the cell response involved measurement of inhibition of cell culture metabolism (mitochondrial succinate dehydrogenase activity) and cell death (release of lactate dehydrogenase). While these three cell types generally ranked metals in ED50 values similarly (V

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

    PubMed

    Feuerborn, Alexander; Möritz, Constanze; Von Bonin, Frederike; Dobbelstein, Matthias; Trümper, Lorenz; Stürzenhofecker, Benjamin; Kube, Dieter

    2006-09-01

    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 derived from cHL are rare and therefore not notably involved in the pathogenesis of the malignant H&RS cells. Re-evaluating the expression in cHL-derived cell lines, we found that in 3/6 of these cell lines, TP53 transcripts are characterized by deletions within exon 4 (L428 cells) and nearly a complete 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 of the transcriptional activity of p53. Transcriptional inactivity was also found for p53 in L428 cells. This study characterizes mutations in TP53 transcripts within cHL cell lines with associated functional defects in the resulting p53 proteins and therefore reintroduces the concept that mutations of TP53 might be involved in the pathogenesis of Hodgkin's lymphoma.

  18. Distinct Small RNA Signatures in Extracellular Vesicles Derived from Breast Cancer Cell Lines

    PubMed Central

    Knutsen, Erik; Nikolaisen, Marlen Aas; Jørgensen, Tor Erik; Johansen, Steinar Daae; Perander, Maria; Seternes, Ole Morten

    2016-01-01

    Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. Understanding the molecular features responsible for this heterogeneity is important for correct diagnosis and better treatment strategies. Extracellular vesicles (EVs) and their associated molecules have gained much attention as players in intercellular communication, ability to precondition specific organs for metastatic invasion, and for their potential role as circulating cancer biomarkers. EVs are released from the cells and contain proteins, DNA, and long and small RNA species. Here we show by high-throughput small RNA-sequencing that EVs from nine different breast cancer cell lines share common characteristics in terms of small RNA content that are distinct from their originating cells. Most strikingly, a highly abundant small RNA molecule derived from the nuclear 28S rRNA is vastly enriched in EVs. The miRNA profiles in EVs correlate with the cellular miRNA expression pattern, but with a few exceptions that includes miR-21. This cancer-associated miRNA is retained in breast cancer cell lines. Finally, we report that EVs from breast cancer cell lines cluster together based on their small RNA signature when compared to EVs derived from other cancer cell lines. Altogether, our data demonstrate that breast cancer cell lines manifest a specific small RNA signature in their released EVs. This opens up for further evaluation of EVs as breast cancer biomarkers. PMID:27579604

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

    PubMed

    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.

  20. Cytogenetic characterization of three cell lines derived from primary cervical tumors of different histologic grade

    SciTech Connect

    Hann, E.; Beauregard, L.; Mikumo, R.

    1994-09-01

    Braum et al.(1993) established three cell lines from keratinizing and nonkeratinizing cervical carcinomas. These cell lines were subsequently analyzed for growth properties and the physical state of the human papillomavirus type 16 genome. TC140, derived from a keratinizing cervical tumor, contains human papillomavirus type 16 in the episomal state. TC-146A and TC-146B, derived from a nonkeratinizing large-cell cervical carcinoma, contain human papillomavirus type 16 in the integrated state. The goal of the present study was to cytogenetically characterize these cell lines, developed from cervical carcinoma with a defined histopathology, in order to shed additional light on the biological basis of the histological and clinical heterogeneity of cervical cancers. Information on solid tumors has been limited because they are often difficult to culture and the karyotypes on the available metaphases are often complex with unidentifiable markers. The chromosomes of these three cell lines were characterized in the present study using GTG-banding. For cell line 140, the most striking chromosomal abnormalities noted were the presence of an i(5p) or i(12p) marker, an isochromosome 8q marker and multiple copies of chromosome 9. For cell line 146A, the most notable chromosomal abnormalities noted were the presence of a marker chromosome 7 with additional materials present on the long arms, an isochomosome of the long arms of chromosome 8 and a question of chromosome 19 markers. For cell line 146B, the most notable chromosomal abnormalities were found to be a deleted X chromosome, a marker chromosome 7 with additional material on the long arm, an isochromosome 8q marker, and isochromosome 16q marker and one or more copies of an isochromosome 17q marker. Fluorescent in situ hybridization experiments performed using select probes further corroborate the results of the above-mentioned conventional cytogenetic studies.

  1. Exact solutions of the derivative nonlinear Schrödinger equation for a nonlinear transmission line.

    PubMed

    Kengne, E; Liu, W M

    2006-02-01

    We consider the derivative nonlinear Schrödinger equation with constant potential as a model for wave propagation on a discrete nonlinear transmission line. This equation can be derived in the small amplitude and long wavelength limit using the standard reductive perturbation method and complex expansion. We construct some exact soliton and elliptic solutions of the mentioned equation by perturbation of its Stokes wave solutions. We find that for some values of the coefficients of the equation and for some parameters of solutions, the graphical representations show some kinds of symmetries such as mirror symmetry and rotational symmetry.

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

    PubMed

    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.

  3. Derivation of Huntington Disease affected Genea017 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea017 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 40 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, genetic analysis confirmed a 46, XY karyotype and male allele pattern through CGH and STR analysis. The hESC line had pluripotent cell morphology, 87% of cells expressed Nanog, 95% Oct4, 88% Tra1-60 and 99% SSEA4, gave a PluriTest pluripotency score of 34.74, novelty of 1.27, demonstrated alkaline phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

  4. Transplantation of a cell line derived from a canine benign mixed mammary tumour into nude mice.

    PubMed

    Priosoeryanto, B P; Tateyama, S; Yamaguchi, R; Uchida, K

    1995-11-01

    The MCM-B2 canine mammary cell line was serially transplanted into nude mice. The tumour masses consisted of elongated pleomorphic cells of varying size in the first to third passages; oval cells, becoming rounder, in the sixth to eighth passages; and cord-like, glandular and duct-like structures with compact radiating projections in the ninth and tenth passages. Ultrastructural and immunohistochemical examination of round cells confirmed their epithelial cell nature, but the morphology of the elongated and oval cells was identical with that of the original cell line. The findings suggest that the MCM-B2 cell line is a multipotential stem cell or is derived from glandular differentiation of mammary gland.

  5. Derivation of FSHD1 affected human embryonic stem cell line Genea049.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea049 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying a deletion in 4q35 with only 5 D4Z4 repeats by PGD linkage analysis, indicative of FSHD1. 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, 90% of cells expressed Nanog, 96% Oct4, 80% Tra1-60 and 99% SSEA4, gave a Pluritest Pluripotency score of 23.16, Novelty of 1.43 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  6. Generation and characterization of bovine bone marrow-derived macrophage cell line.

    PubMed

    Xiao, Jiajia; Xie, Rongxia; Li, Qiaoqiao; Chen, Wuju; Zhang, Yong

    2016-05-01

    Macrophages, as the forefront of innate immune defense, have an important role in the host responses to mycobacterial infection. Therefore, a stable macrophage cell line is needed for future bovine immune system research on the bacterial infection. In this study, we established a bovine macrophage cell line by introducing the human telomerase reverse transcriptase (hTERT) gene into bovine bone marrow-derived macrophages (bBMMs). The TERT-bBMMs cells expressed macrophage surface antigen (CD11b, CD282) and upregulated expression of the cytokines IL-1β, IL-6, IL-10, IL-12, TNF-α in response to bacterial invasion. These results demonstrate that this cell line provide reliable cell model system for future studies on interactions between the bovine macrophages and Mycobacterium tuberculosis.

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

    PubMed

    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.

  8. Molecular, phenotypic, and sample-associated data to describe pluripotent stem cell lines and derivatives

    PubMed Central

    Daily, Kenneth; Ho Sui, Shannan J.; Schriml, Lynn M.; Dexheimer, Phillip J.; Salomonis, Nathan; Schroll, Robin; Bush, Stacy; Keddache, Mehdi; Mayhew, Christopher; Lotia, Samad; Perumal, Thanneer M.; Dang, Kristen; Pantano, Lorena; Pico, Alexander R.; Grassman, Elke; Nordling, Diana; Hide, Winston; Hatzopoulos, Antonis K.; Malik, Punam; Cancelas, Jose A.; Lutzko, Carolyn; Aronow, Bruce J.; Omberg, Larsson

    2017-01-01

    The use of induced pluripotent stem cells (iPSC) derived from independent patients and sources holds considerable promise to improve the understanding of development and disease. However, optimized use of iPSC depends on our ability to develop methods to efficiently qualify cell lines and protocols, monitor genetic stability, and evaluate self-renewal and differentiation potential. To accomplish these goals, 57 stem cell lines from 10 laboratories were differentiated to 7 different states, resulting in 248 analyzed samples. Cell lines were differentiated and characterized at a central laboratory using standardized cell culture methodologies, protocols, and metadata descriptors. Stem cell and derived differentiated lines were characterized using RNA-seq, miRNA-seq, copy number arrays, DNA methylation arrays, flow cytometry, and molecular histology. All materials, including raw data, metadata, analysis and processing code, and methodological and provenance documentation are publicly available for re-use and interactive exploration at https://www.synapse.org/pcbc. The goal is to provide data that can improve our ability to robustly and reproducibly use human pluripotent stem cells to understand development and disease. PMID:28350385

  9. Electrosensory ampullary organs are derived from lateral line placodes in cartilaginous fishes.

    PubMed

    Gillis, J Andrew; Modrell, Melinda S; Northcutt, R Glenn; Catania, Kenneth C; Luer, Carl A; Baker, Clare V H

    2012-09-01

    Ampullary organ electroreceptors excited by weak cathodal electric fields are used for hunting by both cartilaginous and non-teleost bony fishes. Despite similarities of neurophysiology and innervation, their embryonic origins remain controversial: bony fish ampullary organs are derived from lateral line placodes, whereas a neural crest origin has been proposed for cartilaginous fish electroreceptors. This calls into question the homology of electroreceptors and ampullary organs in the two lineages of jawed vertebrates. Here, we test the hypothesis that lateral line placodes form electroreceptors in cartilaginous fishes by undertaking the first long-term in vivo fate-mapping study in any cartilaginous fish. Using DiI tracing for up to 70 days in the little skate, Leucoraja erinacea, we show that lateral line placodes form both ampullary electroreceptors and mechanosensory neuromasts. These data confirm the homology of electroreceptors and ampullary organs in cartilaginous and non-teleost bony fishes, and indicate that jawed vertebrates primitively possessed a lateral line placode-derived system of electrosensory ampullary organs and mechanosensory neuromasts.

  10. Characteristics of a thyroid carcinoma cell line derived from spinal metastasis

    PubMed Central

    Zhou, Zhenhua; Li, Yan; Yan, Xu; Wang, Xudong; Chen, Su; Xiao, Jianru

    2016-01-01

    A thyroid carcinoma cell line named THY28 was established through primary culture of the surgical specimens, which were derived from a Chinese patient with spinal metastasis. The cell morphology, growth kinetics, cell cycle, chromosome number, cell capability of migration, tumorigenicity and cytogenetic features of the cell line were investigated. THY28 cells were subcultured in vitro for more than 50 passages with a human karyotype. The modal number of its chromosomes was mainly from 67 to 85. The doubling time of THY28 cells was 56 hours. The histopathological features of xenograft induced by THY28 cells were consistent with the characteristics of thyroid cancer. The biological and molecular properties of THY28 cells were not entirely consistent with those of other thyroid carcinoma cells such as SW579 and TT cells, indicating biological differences between primary and metastatic thyroid carcinoma cell lines. We have established a novel thyroid carcinoma cell line derived from spinal metastasis, which will provide a useful model for biological or therapeutic studies of thyroid carcinoma metastasis. PMID:27811013

  11. Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst.

    PubMed

    Hwang, Woo Suk; Ryu, Young June; Park, Jong Hyuk; Park, Eul Soon; Lee, Eu Gene; Koo, Ja Min; Jeon, Hyun Yong; Lee, Byeong Chun; Kang, Sung Keun; Kim, Sun Jong; Ahn, Curie; Hwang, Jung Hye; Park, Ky Young; Cibelli, Jose B; Moon, Shin Yong

    2004-03-12

    Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.

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

    PubMed Central

    Hidalgo, Alfredo; Monroy, Alberto; Arana, Rosa Ma; Taja, Lucía; Vázquez, Guelaguetza; Salcedo, Mauricio

    2003-01-01

    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. PMID:12659655

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

    NASA Astrophysics Data System (ADS)

    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

  14. On-line UV spectrophotometric analysis for organic chemistry of novel inorganic polymer derived microreactor.

    PubMed

    Cheon, Jin-Ho; Yoon, Tae-Ho; Hong, Lan-Young; Park, Sang-Hee; Kim, Dong-Pyo

    2009-12-01

    The use of microfluidic or lab-on-a-chip system has shown great promise for many applications. We have previously reported fabrication and application of preceramic polymer derived chemically and mechanically stable microfluidic devices in organic synthesis. Even though organic reactions are successfully performed, it is hard to analyze product to evaluate yields without any time delay except for integration of detecting module in the devices. Moreover small sample volume makes it even difficult to analyze sample by conventional analytical tools. Removal of catalyst and by product before analysis is another hurdle in evaluating performance of microrector. In this paper we describe preliminary results for simple on-line (real-time) quantitative analysis of microchemical reaction in preceramic polymer derived microreactor without reconstruction of microreactor or expensive components. A commercial UVNIS spectrophotometer is used to monitor well established Knoevenagel reaction. To evaluate the performance of presented on-line UV/IS monitoring system, UV/IS data are compared with off-line gas chromatography based analysis system.

  15. Establishment and characterization of a new highly metastatic human osteosarcoma cell line derived from Saos2

    PubMed Central

    Du, Lin; Fan, Qiming; Tu, Bing; Yan, Wei; Tang, Tingting

    2014-01-01

    Osteosarcoma is the most common primary malignancy of bone in adolescents and young adults. There is a shortage of tumorigenic and highly metastatic human osteosarcoma cell lines that can be used for metastasis study. Here we establish and characterize a highly metastatic human osteosarcoma cell line that is derived from Saos2 cell line based on bioluminescence. The occasional pulmonary metastatic cells developed from Saos2 were isolated, harvested, characterized and named Saos2-l. The parental Saos2 and Saos2-l cells were further characterized both in vitro and in vivo. Results showed that Saos2-l cells demonstrated increased cell adhesion, migration and invasion compared to the parental Saos2 cells. Conversely, Saos2-l cells grew at a slightly slower rate than that of the parental cells. When injected into nude mice, Saos2-l cells had a greater increase in developing pulmonary metastases compared to the parental Saos2 cells. Further transcriptional profiling analysis revealed that some gene expression were up-regulated or down-regulated in the highly metastatic Saos2-l cells, indicating possible influencing factors of metastasis. Thus, we have established and characterized a highly metastatic human osteosarcoma cell line that should serve as a valuable tool for future investigations on the pathogenesis, metastasis and potential treatments of human osteosarcoma. PMID:25031706

  16. Assessment of testcross performance and genetic diversity of yellow endosperm maize lines derived from adapted x exotic backcrosses.

    PubMed

    Menkir, A; Olowolafe, M O; Ingelbrecht, I; Fawole, I; Badu-Apraku, B; Vroh, B I

    2006-06-01

    Introduction of exotic maize (Zea mays L.) into adapted tropical germplasm may enhance genetic variability and lead to greater progress from selection. The first objective of this study was to determine if yellow endosperm lines derived from adapted x exotic backcrosses contain exotic alleles that are superior to the recurrent adapted parental line for yield and other agronomic traits in tropical environments. Thirteen exotic yellow maize inbred lines were crossed to an adapted orange line (KUSR) and the F1s were backcrossed to KUSR to generate the first backcrosses. Fifty BC1F4 lines derived from these backcrosses and the recurrent parent were crossed to a common inbred tester (L4001) to form testcrosses, which were evaluated at eight environments in Nigeria. Testcrosses of the BC-derived lines differed significantly for grain yield and other agronomic traits. Only two testcrosses yielded significantly less than L4001 x KUSR, with the best 15 testcrosses producing between 289 and 1,056 kg/ha more grain yield than L4001 x KUSR. The best testcrosses were similar to or better than L4001 x KUSR for other agronomic traits. The second objective of this study was to assess the extent of genetic diversity present among the BC-derived lines. We genotyped 46 BC-derived lines including KUSR and L4001 with 10 AFLP primer pairs and found 491 polymorphic fragments. The average allelic diversity of the lines was 0.30 +/- 0.01. The genetic distance of each BC-derived line from KUSR ranged between 0.49 and 0.91. The average genetic distance for all pairs of the BC-derived lines was 0.68 +/- 0.004, varying from 0.34 to 0.92. The increased grain yield and genetic diversity observed in these studies provide evidence that exotic germplasm can contribute new alleles to expand the genetic base of tropical maize and develop high-yielding hybrids.

  17. Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats.

    PubMed

    Yin, Li; Sun, Mingzeng; Ilic, Zoran; Leffert, Hyam L; Sell, Stewart

    2002-02-01

    Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction.

  18. Evaluation of apoptosis-induction by newly synthesized phthalazine derivatives in breast cancer cell lines.

    PubMed

    Arif, Jamal M; Kunhi, Muhammad; Bekhit, Adnan A; Subramanian, Manogaran P; Al-Hussein, Khalid; Aboul-Enein, Hassan Y; Al-Khodairy, Fahad M

    2006-01-01

    Newly synthesized phthalazine derivatives including copper and platinum complexes were evaluated for cytotoxicity in human breast cancer cell lines. The cells were incubated with the compounds (100 microM) for 72 h and cytotoxicity, apoptosis and DNA content were measured by flow cytometery. Our results suggest that the parent (H1-2), copper (C1-2)- and platinum (P1-2)-derivatized compounds were relatively more active in inducing apoptosis and cell killing in both human breast cancer cell lines, MDA-MB-231 cells being the more sensitive. Other compounds showed weak or no response towards these parameters except H-5 causing 40% apoptosis in MDA-MB-231 cells. Addition of copper or platinum in the structures generally reduced the apoptotic potential. Possible roles for structure activity relationships are discussed.

  19. Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors

    PubMed Central

    Hayashi, Masafumi; Tsukiyama, Tomoyuki; Kimura, Koji; Matsuyama, Shuichi; Minami, Naojiro; Yamada, Masayasu; Imai, Hiroshi

    2016-01-01

    Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3⁄4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRβ, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle. PMID:27907214

  20. Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model Mastomys coucha

    PubMed Central

    Hasche, Daniel; Stephan, Sonja; Savelyeva, Larissa; Westermann, Frank; Rösl, Frank

    2016-01-01

    In the present report we describe the establishment of a spontaneous immortalized skin keratinocyte cell line derived from the skin of the multimammate rodent Mastomys coucha. These animals are used in preclinical studies for a variety of human diseases such as infections with nematodes, bacteria and papillomaviruses, especially regarding cutaneous manifestations such as non-melanoma skin cancer. Here we characterize the cells in terms of their origin and cytogenetic features. Searching for genomic signatures, a spontaneous mutation in the splicing donor sequence of Trp53 (G to A transition at the first position of intron 7) could be detected. This point mutation leads to alternative splicing and to a premature stop codon, resulting in a truncated and, in turn, undetectable form of p53, probably contributing to the process of immortalization. Mastomys coucha-derived skin keratinocytes can be used as an in vitro system to investigate molecular and immunological aspects of infectious agent interactions with their host cells. PMID:27533138

  1. Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization.

    PubMed

    Sidhu, Kuldip S; Tuch, Bernard E

    2006-02-01

    Here we describe the first report of three human embryonic stem cell (hESC) clones, hES 3.1, 3.2, and 3.3, derived from the parent line hES3 by sorting of single-cell preparations by flow cytometry. The viability of single-cell preparations before and after cell sorting remained >98%. The hESC were selected by size gating and forward-angle light scatter and were dispersed directly as single cell/ well into 96-well plates containing human fetal fibroblasts as feeder layers. Single stem cell dispersion into 96-well plates was confirmed by using cells from a hES3 line that constitutively expressed green fluorescence protein (eGFP) under similar conditions of flow cytometry. Three clones were obtained from the parent line hES3 -- hES3.1, 3.2, and 3.3 -- and they have been in continuous culture for more than 1 year. The cloning efficiency was less than <0.5%. These hESC clones show normal stem cell characteristics, such as undifferentiated growth, high nucleocytoplasmic ratio, the same karyotype as that of the parent line (46 XX), stem cell surface markers (i.e., SSEA3, SSEA4, OCT4, TRA-1-60, and TRA-1-81), and gene expression for pluripotency (Oct-4 and nanog). They all formed embryoid bodies in suspension cultures, and after seeding in culture plates they showed pluripotency in vitro by forming cell lineages derived from all three germ layers as indicated by expression of the ectodermal marker nestin, the mesodermal marker renin, and the endodermal markers alpha-fetoprotein and GATA6. All clones showed normal expression of alkaline phosphatase activity, a marker of in vitro pluripotency. When hESC clones (1-2 x 10(6) total) were injected into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice under the kidney capsule, all formed teratomas within 6-8 weeks. Analysis of the stem cell surface marker TRA-1-160 by flow cytometry showed nonsignificant (p < 0.05) differences between the clones and the parent line. The clones also differed in their expression

  2. Generation of cloned mice and nuclear transfer embryonic stem cell lines from urine-derived cells

    PubMed Central

    Mizutani, Eiji; Torikai, Kohei; Wakayama, Sayaka; Nagatomo, Hiroaki; Ohinata, Yasuhide; Kishigami, Satoshi; Wakayama, Teruhiko

    2016-01-01

    Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal. PMID:27033801

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

    PubMed

    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.

  4. Mass loss from alpha Cyg /A2Ia/ derived from the profiles of low excitation Fe II lines

    NASA Technical Reports Server (NTRS)

    Hensberge, H.; De Loore, C.; Lamers, H. J. G. L. M.; Bruhweiler, F. C.

    1982-01-01

    The low-excitation Fe II lines in the spectral region 2000-3000 A are studied in the spectrum of alpha-Cyg. The profiles of the resonance lines are described by four representative parameters, and a preliminary model is derived from the dependence of these parameters on theoretical line strength, taking into account the influence of blending photospheric lines in an overall and qualitative way. At least 11% of all iron in the wind is once ionized, unless a non-thermal heating source enhances the fraction Fe(++) without destroying much Al(+). It is shown that the contribution of blending photospheric absorption lines to weaker P Cygni profiles has been previously largely underestimated. The mass loss rate corresponding to the model is derived, and is smaller by a factor of 500 than the one derived from the infrared excess by Barlow and Cohen (1977).

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

    PubMed

    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.

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

    PubMed Central

    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

  7. Establishment of lal-/- myeloid lineage cell line that resembles myeloid-derived suppressive cells.

    PubMed

    Ding, Xinchun; Wu, Lingyan; Yan, Cong; Du, Hong

    2015-01-01

    Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an "MDSC-like" cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an "MDSC-like" cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.

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

    PubMed

    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.

  9. Preferential metabolism of N-nitrosodiethylamine by two cell lines derived from human pulmonary adenocarcinomas

    SciTech Connect

    Falzon, M.; McMahon, J.B.; Gazdar, A.F.; Schuller, H.M.

    1986-01-01

    Diethylnitrosamine (DEN), in common with other nitrosamines, is a carcinogenic agent which produces tumors in a wide variety of tissues in experimental animals. The pulmonary Clara cell is a major target of N-nitrosamine-induced carcinogenesis in hamsters and rats. DEN is believed to require metabolic activation to elicit its carcinogenic effects. The metabolism of (/sup 14/C)DEN was studied in two cell lines derived from human lung adenocarcinomas and two cell lines derived from human small cell lung cancers by monitoring /sup 14/CO/sub 2/ production and covalent binding of radiolabel from (/sup 14/C)DEN to the cell protein and DNA fractions. (/sup 14/C)DEN was metabolized by adenocarcinoma-derived NCI-H322 (with Clara cell features) and NCI-H358 (with features of alveolar type II cells) but not by NCI-H69 and NCI-H128 (derived from small cell carcinoma). Metabolism was markedly inhibited by heat denaturation of the cell protein. (/sup 14/C)DEN metabolism by NCI-H322 was greatly decreased when the incubation was carried out under anaerobic conditions and in the presence of a carbon monoxide enriched atmosphere. These results suggested the involvement of the cytochrome P-450-dependent monooxygenase enzyme system. Metabolism by NCI-H358 was also decreased in the absence of oxygen or presence of carbon monoxide although the effects were relatively small compared with the results with NCI-H322. On the other hand, aspirin or indomethacin, which are inhibitors of the fatty acid cyclooxygenase component of prostaglandin endoperoxide synthetase, preferentially inhibited (/sup 14/C)DEN metabolism by NIC-H358. There were little or no effects of these inhibitors on the metabolism of DEN in NCI-H322. The data suggest that DEN metabolism in different lung cell types may be carried out by different enzyme systems which in turn may contribute to the selective effect of DEN in the lung.

  10. Differential gene expression in human, murine, and cell line-derived macrophages upon polarization.

    PubMed

    Spiller, Kara L; Wrona, Emily A; Romero-Torres, Saly; Pallotta, Isabella; Graney, Pamela L; Witherel, Claire E; Panicker, Leelamma M; Feldman, Ricardo A; Urbanska, Aleksandra M; Santambrogio, Laura; Vunjak-Novakovic, Gordana; Freytes, Donald O

    2016-09-10

    The mechanisms by which macrophages control the inflammatory response, wound healing, biomaterial-interactions, and tissue regeneration appear to be related to their activation/differentiation states. Studies of macrophage behavior in vitro can be useful for elucidating their mechanisms of action, but it is not clear to what extent the source of macrophages affects their apparent behavior, potentially affecting interpretation of results. Although comparative studies of macrophage behavior with respect to cell source have been conducted, there has been no direct comparison of the three most commonly used cell sources: murine bone marrow, human monocytes from peripheral blood (PB), and the human leukemic monocytic cell line THP-1, across multiple macrophage phenotypes. In this study, we used multivariate discriminant analysis to compare the in vitro expression of genes commonly chosen to assess macrophage phenotype across all three sources of macrophages, as well as those derived from induced pluripotent stem cells (iPSCs), that were polarized towards four distinct phenotypes using the same differentiation protocols: M(LPS,IFN) (aka M1), M(IL4,IL13) (aka M2a), M(IL10) (aka M2c), and M(-) (aka M0) used as control. Several differences in gene expression trends were found among the sources of macrophages, especially between murine bone marrow-derived and human blood-derived M(LPS,IFN) and M(IL4,IL13) macrophages with respect to commonly used phenotype markers like CCR7 and genes associated with angiogenesis and tissue regeneration like FGF2 and MMP9. We found that the genes with the most similar patterns of expression among all sources were CXCL-10 and CXCL-11 for M(LPS,IFN) and CCL17 and CCL22 for M(IL4,IL13). Human PB-derived macrophages and human iPSC-derived macrophages showed similar gene expression patterns among the groups and genes studied here, suggesting that iPSC-derived monocytes have the potential to be used as a reliable cell source of human macrophages

  11. Establishment and characterization of a cell line derived from Eptesicus nilssonii

    PubMed Central

    HORIE, Masayuki; AKASAKA, Takumi; MATSUDA, Sachiko; OGAWA, Haruko; IMAI, Kunitoshi

    2016-01-01

    Bats of the genus Eptesicus have several non-retroviral RNA virus-derived sequences in their genomes, among which an endogenous bornavirus-like L element, named eEBLL-1, was suggested to encode functional proteins in the hosts. However, the function of eEBLL-1 remains unclear due to a lack of appropriate investigation tools, such as cultured cells expressing eEBLL-1. Here, we established a continuous cell line, named HAMOI-EnK cells, from kidney of Eptesicus nilssonii. HAMOI-EnK cells are robust and could be passaged for at least 10 months. eEBLL-1 in the genomes of HAMOI-EnK cells retains an intact open reading frame. Additionally, eEBLL-1 is transcribed in the sense-orientation in cells. To our knowledge, this is the first report to demonstrate that eEBLL-1 is transcribed in cultured cells. PMID:27499253

  12. Cabergoline Decreases Alcohol Drinking and Seeking Behaviors Via Glial Cell Line-Derived Neurotrophic Factor

    PubMed Central

    Carnicella, Sebastien; Ahmadiantehrani, Somayeh; He, Dao-Yao; Nielsen, Carsten K.; Bartlett, Selena E.; Janak, Patricia H.; Ron, Dorit

    2010-01-01

    Background Cabergoline is an ergotamine derivative that increases the expression of glial cell line-derived neurotrophic factor (GDNF) in vitro. We recently showed that GDNF in the ventral tegmental area (VTA) reduces the motivation to consume alcohol. We therefore set out to determine whether cabergoline administration decreases alcohol-drinking and -seeking behaviors via GDNF. Methods Reverse transcription polymerase chain reaction (RT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) were used to measure GDNF levels. Western blot analysis was used for phosphorylation experiments. Operant self-administration in rats and a two-bottle choice procedure in mice were used to assess alcohol-drinking behaviors. Instrumental performance tested during extinction was used to measure alcohol-seeking behavior. The [35S]GTPγS binding assay was used to assess the expression and function of the dopamine D2 receptor (D2R). Results We found that treatment of the dopaminergic-like cell line SH-SY5Y with cabergoline and systemic administration of cabergoline in rats resulted in an increase in GDNF level and in the activation of the GDNF pathway. Cabergoline treatment decreased alcohol-drinking and -seeking behaviors including relapse, and its action to reduce alcohol consumption was localized to the VTA. Finally, the increase in GDNF expression and the decrease in alcohol consumption by cabergoline were abolished in GDNF heterozygous knockout mice. Conclusions Together, these findings suggest that cabergoline-mediated upregulation of the GDNF pathway attenuates alcohol-drinking behaviors and relapse. Alcohol abuse and addiction are devastating and costly problems worldwide. This study puts forward the possibility that cabergoline might be an effective treatment for these disorders. PMID:19232578

  13. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    SciTech Connect

    Youakim, A.; Herscovics, A.

    1985-11-01

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-(2-TH)mannose or L-(5,6-TH)fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with (2-TH)mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with (2-TH)mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-(1,6-TH)glucosamine and L-(1- UC)fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced TH-labeled N-acetylglucosamine and N-acetylgalactosamine.

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

    PubMed

    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.

  15. A retrovirus isolated from cell lines derived from neurofibromas in bicolor damselfish (Pomacentrus partitus).

    PubMed

    Schmale, M C; Aman, M R; Gill, K A

    1996-06-01

    Damselfish neurofibromatosis (DNF) is a naturally occurring, neoplastic disease affecting bicolor damselfish (Pomacentrus partitus) living on coral reefs in southern Florida, USA. The disease consists of multiple neurofibromas, neurofibrosarcomas and chromatophoromas and has been proposed as an animal model for neurofibromatosis type 1 in humans. DNF is transmissible by injection of crude tumour homogenates, cell-free filtrates of homogenates or cells from tumour cell lines. An analysis of tumorigenic cell lines derived from fish with spontaneous or experimentally induced DNF revealed virus particles budding from cells and present in conditioned media. The 90-110 nm particles resembled type C retroviruses. This virus exhibited a buoyant density of 1.14-1.17 g/cm2 in sucrose, at least six virus proteins of 15 to 80 kDa and reverse transcriptase (RT) activity. RT activity was maximized with a poly(rC).oligo(dG) template.primer combination and Mn2+ at a concentration of 0.5-1.0 mM. The optimum temperature for RT was determined to be 20 degrees C, a finding consistent with the ambient temperatures encountered by this species. This retrovirus, tentatively named damselfish neurofibromatosis virus (DNFV) may be the aetiological agent of DNF. Whether DNFV or another, as yet unidentified, virus is the cause of DNF, this agent may be unique in virus oncogenesis; neoplastic transformation of the cell types involved in DNF, Schwann cells and chromatophores, has not been documented in any other transmissible tumour.

  16. Densities, temperatures, pressures, and abundances derived from O II recombination lines in H II regions and their implications

    SciTech Connect

    Peimbert, Antonio; Peimbert, Manuel E-mail: peimbert@astro.unam.mx

    2013-12-01

    Based on high-quality observations of multiplet V1 of O II and the NLTE atomic computations of O II, we study the density and temperature of a sample of H II regions. We find that the signature for oxygen-rich clumps of high density and low temperature is absent in all objects of our sample: one extragalactic and eight Galactic H II regions. The temperatures derived from (1) recombination lines (RLs) of O II, and (2) RLs of H I together with Balmer continua are lower than those derived from forbidden lines, while the densities derived from RLs of O II are similar or smaller than densities derived from forbidden lines. Electron pressures derived from collisionally excited lines are about two times larger than those derived from RLs. These results imply that the proper abundances are those derived from RLs and suggest that other processes in addition to direct photoionization, such as dissipation of turbulent energy in shocks, magnetic reconnection, and shadowed regions, might be responsible for the large abundance discrepancy factor and t {sup 2} values observed in H II regions.

  17. Tumorigenicity, invasion and metastasis of the small cell lung cancer cell line NCI-H69 and two derivative lines MOG-H69V and MOG-H69VZ.

    PubMed

    Khan, M Z; McNicol, A M; Freshney, R I

    1996-01-01

    Two adherent cell lines MOG-H69V and MOG-H69VZ have been isolated from a continuous cell line, NCI-H69, derived from human small cell lung cancer by Carney et al, [1987]. They have been established and characterised morphologically, biochemically, and for growth characteristics in vitro Khan et al (19). In the present study both the parental and the derivative lines have been investigated for invasiveness in vitro and in vivo. The parental line showed an early invasiveness compared with both the derivative cell lines. All cell lines formed tumours in nude mice with 100% take rate. Xenograft histology of all the cell lines revealed pleomorphic tumours, however the derivative lines showed areas of focal, large, spindle cells containing both acidic and neutral mucin, and spaces between the cells were found filled with alcianophilic, amorphous material. The parental line was invasive and metastatic. Tumours of both the derivative lines were non-metastatic under similar conditions. They were also investigated for neuroendocrine-cell marker expression. These data show that while the behaviour of the parental line was compatible with small cell lung cancer, that of the derivative lines was more indicative of non-small cell lung cancer, both in vitro and in vivo. As previous data suggested a common origin of the parental and the derivative lines, probably from a stem cell subpopulation present in the parental line, these lines represent a useful model for the study of phenotypic changes in lung cancer.

  18. Multiple Breast Cancer Cell-Lines Derived from a Single Tumor Differ in Their Molecular Characteristics and Tumorigenic Potential

    PubMed Central

    Mosoyan, Goar; Nagi, Chandandeep; Marukian, Svetlana; Teixeira, Avelino; Simonian, Anait; Resnick-Silverman, Lois; DiFeo, Analisa; Johnston, Dean; Reynolds, Sandra R.; Roses, Daniel F.; Mosoian, Arevik

    2013-01-01

    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 Fuorescent 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 identify mechanisms

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:25061293

  1. Derivation of human embryonic stem cell lines from biopsied blastomeres on human feeders with minimal exposure to xenomaterials.

    PubMed

    Ilic, Dusko; Giritharan, Gnanaratnam; Zdravkovic, Tamara; Caceres, Eduardo; Genbacev, Olga; Fisher, Susan J; Krtolica, Ana

    2009-11-01

    In a continuous effort to improve the generation of therapeutic grade human embryonic stem cell (hESC) lines, we focused on preserving developmental capacity of the embryos, minimizing the exposure to xenomaterials, increasing derivation efficacy, and reducing the complexity of the derivation procedure. In this study, we describe an improved method for efficient derivation of hESC lines from blastomeres of biopsied embryos. Our protocol substituted feeder cells of mouse origin with human foreskin fibroblasts (HFFs), limited serum exposure of cells to formation of the initial outgrowth, and increased derivation efficacy from 12.5% (one hESC line out of 13 biopsies) to 50% (3 out of 6 biopsies) by using early population doubling (PD) HFFs. In addition, it eliminated a need for embryo-blastomere coculture, thus reducing the complexity of the culture and enabling continued development of the biopsied embryo under optimal conditions. All derived lines maintained normal karyotype and expressed totipotent phenotype including the ability to differentiate into trophectoderm and all three germ layers.

  2. Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity

    PubMed Central

    Kruger, Matthew; Boney, Robert; Ordoobadi, Alexander J.; Sommers, Thomas F.; Trapani, Josef G.; Coffin, Allison B.

    2016-01-01

    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. PMID:27065807

  3. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    SciTech Connect

    Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z. . E-mail: hzsheng2003@yahoo.com

    2006-11-01

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.

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

    PubMed

    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.

  5. Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

    PubMed

    Morel, G R; Sosa, Y E; Bellini, M J; Carri, N G; Rodriguez, S S; Bohn, M C; Goya, R G

    2010-05-19

    Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.

  6. Synthesis of N-methylarylnitrones derived from alkyloxybenzaldehydes and antineoplastic effect on human cancer cell lines.

    PubMed

    Costa, Débora S S; Martino, Thiago; Magalhães, Fernanda C; Justo, Graça; Coelho, Marsen G P; Barcellos, Julio C F; Moura, Victor B; Costa, Paulo R R; Sabino, Kátia C C; Dias, Ayres G

    2015-05-01

    New O-isoprenylated-N-methylarylnitrones derived from isomeric o, m and p-hydroxybenzaldehydes have been prepared and the antineoplastic effects on human cancer cell lines were evaluated. The O-geranylated nitrone LQB-278 (1b) and its isomers 2b and 3b inhibited the NO production, but the anti-leukemic activity was drastically dependent on nitrone isomer, with the 1b being the most effective one (IC₅₀ of 6.7 μM) on Jurkat leukemia cell, by MTT assay. In addition, 1b up-regulated p21CIP1/WAF1/Sdi1 protein expression (flow cytometry), a cell cycle inhibitor, reduced cell growth, and induced DNA fragmentation (increased sub-G1 phase cells) and phosphatidylserine externalization in plasmatic membrane (increased annexin V positive cells). Finally, the 1b up-regulation of p21 expression and apoptosis induction seem to be the mechanisms by which it promotes its anti-leukemic effects, making this new molecular architecture a promising prototype for leukemia intervention.

  7. Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines

    PubMed Central

    Kittrell, Frances; Valdez, Kelli; Elsarraj, Hanan; Hong, Yan; Medina, Daniel; Behbod, Fariba

    2016-01-01

    The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al., 2009; Valdez et al., 2011). In order to demonstrate that transplantation procedure is successful, the transplanted mammary glands may be excised as early as two weeks following intraductal injection of cells followed by Hematoxylin and Eosin (H&E) staining and/or immunofluorescence staining using human specific cytokeratin 5 and/or 19 [please see Figures 2–4 in Behbod et al. (2009)]. Additionally, the presence of trypan blue inside the mouse mammary ducts immediately following intraductal injection is the best indicator that the injection was successful (Video 1 starting at 4:33 sec). PMID:27446983

  8. Photodynamic effects of a novel pterin derivative on a pancreatic cancer cell line

    SciTech Connect

    Yamada, Hiroko; Arai, Toshiyuki . E-mail: arai@kuhp.kyoto-u.ac.jp; Endo, Nobuyuki; Yamashita, Kouhei; Nonogawa, Mitsuru; Makino, Keisuke; Fukuda, Kazuhiko; Sasada, Masataka; Uchiyama, Takashi

    2005-08-05

    6-Formylpterin (6FP) has the potential to produce singlet oxygen ({sup 1}O{sub 2}) under UV-A radiation. In order to apply this potential to anti-cancer photodynamic therapy (PDT), we prepared a novel variant of 6FP, 2-(N,N-dimethylaminomethyleneamino)-6-formyl-3-pivaloylpteridine-4-one (6FP-tBu-DMF), and examined its photodynamic effects on a pancreatic cancer cell line, Panc-1 cells. The study using laser scanning confocal microscopy showed that the drug uptake, the {sup 1}O{sub 2} generation, and cell death were observed in the 6FP-tBu-DMF-treated cells, while these phenomena were not observed in the 6FP-treated cells. The MTT assay also showed the decrease in cell viability only in the 6FP-tBu-DMF-treated cells. Since 6FP and 6FP-tBu-DMF generate {sup 1}O{sub 2} to the same extent under UV-A radiation in aqueous solutions, these results indicated that the differences in the photodynamic effects between 6FP and 6FP-tBu-DMF were entirely attributed to the differences in the cell permeability between them. The development of cell permeable pterin derivatives has the potential for application in PDT.

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

    PubMed

    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.

  10. Comparative susceptibilities of insect cell lines to infection by the occlusion-body derived phenotype of baculoviruses.

    PubMed

    Lynn, Dwight E

    2003-07-01

    Twelve insect cell lines from six species were tested for susceptibility to baculovirus infection by occlusion-derived virus (ODV) phenotype through the use of a typical endpoint assay procedure. ODV from three nucleopolyhedroviruses were prepared by alkali treatment (sodium carbonate) of occlusion bodies (OBs) and the virus preparations were titered on various cell lines. More than a four-log difference was realized for each of theses viruses between the various cell lines. The TN368 line from Trichoplusia ni was only marginally susceptible to ODV from each virus, showing only 3-6 infectious units (IU) per million OBs while the gypsy moth line, LdEp was most susceptible, realizing more than 100,000 IU/million OBs. The other lines tested showed various levels of susceptibility between these two extremes and also varied between the three viruses tested. In additional tests, the ODV were treated with trypsin prior to application to the cells. With most cell lines, this treatment increased the infectivity of each virus by 2-10-fold. Exceptions to this trend included the gypsy moth LdEp line, on which the trypsinized ODV from two of the viruses were slightly less infectious than each virus without trypsin, and the TN-368 line, on which the trypsinized ODV was 5,000-75,000 times more infectious. The variable results of trypsinized virus on the different lines are probably due to the levels of endogenous protease activity in the various lines, but the mode of action of the trypsin has not been elucidated. Ultimately, the variable response of cell lines to ODV of different viruses, and the variable effects of trypsin on the ODV may lead to an improved understanding of the infection process of this virus phenotype as well as factors relating to baculovirus host range.

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Identification of QTL conditioning partial resistance to white mold in kidney bean line VA19 derived from an interspecific population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scarlet-runner bean (Phaseolus coccineus L.), a representative species of the secondary gene pool of common bean, is a potential source of white mold resistance for improving dry bean and snap bean. VA19 is a light-red kidney bean line that possesses resistance to white mold putatively derived from...

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

    PubMed

    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.

  14. Differential expression of a subset of ribosomal protein genes in cell lines derived from human nasopharyngeal epithelium.

    PubMed

    Sim, Edmund Ui Hang; Ang, Chow Hiang; Ng, Ching Ching; Lee, Choon Weng; Narayanan, Kumaran

    2010-02-01

    Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.

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

    PubMed

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

    2014-10-08

    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.

  16. Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

    PubMed Central

    Paris, Irmgard; Muñoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

    2011-01-01

    Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50μM), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100μM), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10μM vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30μM aminochrome in the absence and presence of DIC, respectively, whereas 10μM rapamycin preincubated 24 h before addition of 50μM aminochrome in the absence and the presence of 100μM DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

  17. Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines

    SciTech Connect

    Bronzert, D.A.; Pantazis, P.; Antoniades, H.N.; Kasid, A.; Davidson, N.; Dickson, R.B.; Lippman, M.E.

    1987-08-01

    The authors report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or competence activity that is capable of inducing incorporation of (/sup 3/H) thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95/sup 0/C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approx. =30 kDa on NaDodSO/sub 4//polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO/sub 4//polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth.

  18. Preclinical testing of selective Aurora kinase inhibitors on a medullary thyroid carcinoma-derived cell line.

    PubMed

    Tuccilli, Chiara; Baldini, Enke; Prinzi, Natalie; Morrone, Stefania; Sorrenti, Salvatore; Filippini, Angelo; Catania, Antonio; Alessandrini, Stefania; Rendina, Roberta; Coccaro, Carmela; D'Armiento, Massimino; Ulisse, Salvatore

    2016-05-01

    Deregulated expression of the Aurora kinases (Aurora-A, B, and C) is thought to be involved in cell malignant transformation and genomic instability in several cancer types. Over the last decade, a number of small-molecule inhibitors of Aurora kinases have been developed, which have proved to efficiently restrain malignant cell growth and tumorigenicity. Regarding medullary thyroid carcinoma (MTC), we previously showed the efficacy of a pan-Aurora kinase inhibitor (MK-0457) in impairing growth and survival of the MTC-derived cell line TT. In the present study, we sought to establish if one of the Aurora kinases might represent a preferential target for MTC therapy. The effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on TT cell proliferation, apoptosis, cell cycle, and ploidy. The two inhibitors reduced TT cell proliferation in a time- and dose-dependent manner, with IC50 of 19.0 ± 2.4 nM for MLN8237 and 401.6 ± 44.1 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited phosphorylation of histone H3 (Ser10) by Aurora-B, while it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Cytofluorimetry experiments showed that both inhibitors induced accumulation of cells in G2/M phase and increased the subG0/G1 fraction and polyploidy. Finally, both inhibitors triggered apoptosis. We demonstrated that inhibition of either Aurora-A or Aurora-B has antiproliferative effects on TT cells, and thus it would be worthwhile to further investigate the therapeutical potential of Aurora kinase inhibitors in MTC treatment.

  19. Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor.

    PubMed

    Aponte, Pedro M; Soda, Takeshi; van de Kant, H J G; de Rooij, Dirk G

    2006-06-01

    Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A(s)) and paired (A(pr)) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A(s) to A(pr) and spermatogonial chains (A(al)) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.

  20. Elucidating the cancer-specific genetic alteration spectrum of glioblastoma derived cell lines from whole exome and RNA sequencing

    PubMed Central

    Somasundaram, Kumaravel

    2015-01-01

    Cell lines derived from tumor tissues have been used as a valuable system to study gene regulation and cancer development. Comprehensive characterization of the genetic background of cell lines could provide clues on novel genes responsible for carcinogenesis and help in choosing cell lines for particular studies. Here, we have carried out whole exome and RNA sequencing of commonly used glioblastoma (GBM) cell lines (U87, T98G, LN229, U343, U373 and LN18) to unearth single nucleotide variations (SNVs), indels, differential gene expression, gene fusions and RNA editing events. We obtained an average of 41,071 SNVs out of which 1,594 (3.88%) were potentially cancer-specific. The cell lines showed frequent SNVs and indels in some of the genes that are known to be altered in GBM- EGFR, TP53, PTEN, SPTA1 and NF1. Chromatin modifying genes- ATRX, MLL3, MLL4, SETD2 and SRCAP also showed alterations. While no cell line carried IDH1 mutations, five cell lines showed hTERT promoter activating mutations with a concomitant increase in hTERT transcript levels. Five significant gene fusions were found of which NUP93-CYB5B was validated. An average of 18,949 RNA editing events was also obtained. Thus we have generated a comprehensive catalogue of genetic alterations for six GBM cell lines. PMID:26496030

  1. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  2. Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies.

    PubMed

    Hartman, Taymar E; Sar, Nalin; Genereux, Kimberly; Barritt, Diana S; He, Yimin; Burky, John E; Wesson, Mark C; Tso, J Yun; Tsurushita, Naoya; Zhou, Weichang; Sauer, Paul W

    2007-02-01

    Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg

  3. Heterogeneity of cell lines derived after transformation of early passage rodent cells by the Ha-ras1 human oncogene.

    PubMed

    Spandidos, D A; Freshney, M; Wilkie, N M

    1985-01-01

    The chromosome patterns of Chinese hamster cell lines derived after immortalization or tumorigenic conversion of early passage cells with recombinants carrying the mutated T24 or the normal human Ha-ras1 gene have been characterized by trypsin-Giemsa banding. Whereas immortalized Chinese hamster cell lines exhibited a near normal karyotype, tumorigenic cell lines were found to have abnormal karyotypes carrying marker chromosomes. Moreover, chromosomal patterns correlated with growth in semisolid media and tumourigenicity in nude mice. Similarly, malignant conversion of early passage Syrian hamster cells, with a recombinant carrying the mutated T24 human Ha-ras1 gene, resulted in cells with a near diploid karyotype. On the other hand, tumorigenic conversion of early passage Wistar rat cells with the same oncogene produced cell lines with heteroploid karyotypes. More chromosomal alterations have been observed during further growth of these cells. It is suggested that the transformed phenotype in these cells may be dependent on the chromosomal instability.

  4. Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.

    PubMed Central

    Hoshino, H; Esumi, H; Miwa, M; Shimoyama, M; Minato, K; Tobinai, K; Hirose, M; Watanabe, S; Inada, N; Kinoshita, K; Kamihira, S; Ichimaru, M; Sugimura, T

    1983-01-01

    By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro. Images PMID:6193528

  5. Derivation of the physical parameters for strong and weak flares from the Hα line

    NASA Astrophysics Data System (ADS)

    Semeida, M. A.; Rashed, M. G.

    2016-06-01

    The two flares of 19 and 30 July 1999 were observed in the Hα line using the multichannel flare spectrograph (MFS) at the Astronomical Institute in Ondřejov, Czech Republic. We use a modified cloud method to fit the Hα line profiles which avoids using the background profile. We obtain the four parameters of the two flares: the source function, the optical thickness at line center, the line-of-sight velocity and the Doppler width. The observed asymmetry profiles have been reproduced by the theoretical ones based on our model. A discussion is made about the results of strong and weak flares using the present method.

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

    PubMed

    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.

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

    PubMed Central

    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

  8. Development and characterization of two porcine monocyte-derived macrophage cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell lines Cdelta2+ and Cdelta2- were developed from monocytes obtained from a 10-month-old, crossbred, female pig. These cells morphologically resembled macrophages, stained positively for a-naphthyl esterase and negatively for peroxidase. The cell lines were bactericidal and highly phagocytic. ...

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

    PubMed

    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.

  10. Uniformity under in vitro conditions: Changes in the phenotype of cancer cell lines derived from different medulloblastoma subgroups

    PubMed Central

    Chlapek, Petr; Zitterbart, Karel; Kren, Leos; Filipova, Lenka; Sterba, Jaroslav; Veselska, Renata

    2017-01-01

    Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures. PMID:28231263

  11. Generation of spinocerebellar ataxia type 2 patient-derived iPSC line H266.

    PubMed

    Marthaler, Adele G; Schmid, Benjamin; Tubsuwan, Alisa; Poulsen, Ulla B; Hyttel, Poul; Nielsen, Troels T; Nielsen, Jørgen E; Holst, Bjørn

    2016-01-01

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease primarily affecting the cerebellum. Very little is known about the molecular mechanisms underlying the disease and, to date, no cure or treatment is available. Here, we demonstrate the generation of an induced pluripotent stem cell (iPSC) line of a SCA2 patient. The selected clone has been proven to be a bona fide iPSC line, which retains a normal karyotype. Due to its differentiation potential into neurons, this iPSC line will be a valuable tool in studying a disease-specific phenotype of SCA2.

  12. Generation of spinocerebellar ataxia type 2 patient-derived iPSC line H271.

    PubMed

    Marthaler, Adele G; Tubsuwan, Alisa; Schmid, Benjamin; Poulsen, Ulla B; Hyttel, Poul; Nielsen, Jørgen E; Nielsen, Troels T; Holst, Bjørn

    2016-01-01

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease primarily affecting the cerebellum. Very little is known about the molecular mechanisms underlying the disease and, to date, no cure or treatment is available. Here, we demonstrate the generation of an induced pluripotent stem cell (iPSC) line of a SCA2 patient. The selected clone has been proven to be a bona fide iPSC line, which retains a normal karyotype. Due to its differentiation potential into neurons, this iPSC line will be a valuable tool in studying a disease-specific phenotype of SCA2.

  13. Generation of spinocerebellar ataxia type 2 patient-derived iPSC line H196.

    PubMed

    Marthaler, Adele G; Schmid, Benjamin; Tubsuwan, Alisa; Poulsen, Ulla B; Hyttel, Poul; Nielsen, Troels T; Nielsen, Jørgen E; Holst, Bjørn

    2016-01-01

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease primarily affecting the cerebellum. Very little is known about the molecular mechanisms underlying the disease and, to date, no cure or treatment is available. Here, we demonstrate the generation of an induced pluripotent stem cell (iPSC) line of a SCA2 patient. The selected clone has been proven to be a bona fide iPSC line, which retains a normal karyotype. Due to its differentiation potential into neurons, this iPSC line will be a valuable tool in studying a disease-specific phenotype of SCA2.

  14. Novel imidazole derivatives as heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2) inhibitors and their cytotoxic activity in human-derived cancer cell lines.

    PubMed

    Salerno, Loredana; Pittalà, Valeria; Romeo, Giuseppe; Modica, Maria N; Marrazzo, Agostino; Siracusa, Maria A; Sorrenti, Valeria; Di Giacomo, Claudia; Vanella, Luca; Parayath, Neha N; Greish, Khaled

    2015-01-01

    Heme oxygenase (HO) is a cytoprotective enzyme that can be overexpressed in some pathological conditions, including certain cancers. In this work, novel imidazole derivatives were designed and synthesized as inhibitors of heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). In these compounds the imidazole ring, crucial for the activity, is connected to a hydrophobic group, represented by aryloxy, benzothiazole, or benzoxazole moieties, by means of alkyl or thioalkyl chains of different length. Many of the tested compounds were potent and/or selective against one of the two isoforms of HO. Furthermore, most of the pentyl derivatives showed to be better inhibitors of HO-2 with respect to HO-1, revealing a critical role of the alkyl chain in discriminating between the two isoenzymes. Compounds which showed the better profile of HO inhibition were selected and tested to evaluate their cytotoxic properties in prostate and breast cancer cell lines (DU-145, PC3, LnCap, MDA-MB-231, and MCF-7). In these assays, aryloxyalkyl derivatives resulted more cytotoxic than benzothiazolethioalkyl ones; in particular compound 31 was active against all the cell lines tested, confirming the anti-proliferative properties of HO inhibitors and their potential use in the treatment of specific cancers.

  15. Design, synthesis, and antiproliferative activity of 3,4-diarylpyrazole-1-carboxamide derivatives against melanoma cell line.

    PubMed

    El-Gamal, Mohammed I; Choi, Hong Seok; Cho, Hae-Guk; Hong, Jun Hee; Yoo, Kyung Ho; Oh, Chang-Hyun

    2011-11-01

    Synthesis of a new series of 3,4-diarylpyrazole-1-carboxamide derivatives is described. Their antiproliferative activity against A375P human melanoma cell line was tested and the effect of substituents on the diarylpyrazole scaffold was investigated. The biological results indicated that five synthesized compounds (Ig, Ii, IIc, IIg, and IIh) exhibited similar activity to Sorafenib. In addition, three compounds (IIa, IIb, and IIi) were more potent than Sorafenib. Among all of these derivatives, compound IIa which has dimethylamino and phenolic moieties showed the most potent antiproliferative activity against A375P human melanoma cell line. Virtual screening was carried out through docking of the most potent compound IIa into the domain of V600E-b-Raf and the binding mode was studied.

  16. Relative accuracy of grid references derived from postcode and address in UK epidemiological studies of overhead power lines.

    PubMed

    Swanson, J; Vincent, T J; Bunch, K J

    2014-12-01

    In the UK, the location of an address, necessary for calculating the distance to overhead power lines in epidemiological studies, is available from different sources. We assess the accuracy of each. The grid reference specific to each address, provided by the Ordnance Survey product Address-Point, is generally accurate to a few metres, which will usually be sufficient for calculating magnetic fields from the power lines. The grid reference derived from the postcode rather than the individual address is generally accurate to tens of metres, and may be acceptable for assessing effects that vary in the general proximity of the power line, but is probably not acceptable for assessing magnetic-field effects.

  17. Establishment of an ES cell-derived murine megakaryocytic cell line, MKD1, with features of primary megakaryocyte progenitors.

    PubMed

    Chagraoui, Hedia; Porcher, Catherine

    2012-01-01

    Because of the scarcity of megakaryocytes in hematopoietic tissues, studying megakaryopoiesis heavily relies on the availability of appropriate cellular models. Here, we report the establishment of a new mouse embryonic stem (ES) cell-derived megakaryocytic cell line, MKD1. The cells are factor-dependent, their cell surface immunophenotype and gene expression profile closely resemble that of primary megakaryocyte progenitors (MkPs) and they further differentiate along the megakaryocyte lineage upon valproic acid treatment. At a functional level, we show that ablation of SCL expression, a transcription factor critical for MkP maturation, leads to gene expression alterations similar to that observed in primary, Scl-excised MkPs. Moreover, the cell line is amenable to biochemical and transcriptional analyses, as we report for GpVI, a direct target of SCL. Thus, the MKD1 cell line offers a pertinent experimental model to study the cellular and molecular mechanisms underlying MkP biology and more broadly megakaryopoiesis.

  18. Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

    SciTech Connect

    Gu Yongpeng; Li Hongzhen; Miki, Jun; Kim, Kee-Hong; Furusato, Bungo; Sesterhenn, Isabell A.; Chu, Wei-Sing; McLeod, David G.; Srivastava, Shiv; Ewing, Charles M.; Isaacs, William B.; Rhim, Johng S. . E-mail: jrhim@cpdr.org

    2006-04-01

    In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.

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

    PubMed

    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.

  20. [Comparative characteristics of new mesenchymal stem cell lines derived from human embryonic stem cells, bone marrow and foreskin].

    PubMed

    Krylova, T A; Kol'tsova, A M; Zenin, V V; Musorina, A S; Iakovleva, T K; Polianskaia, G G

    2012-01-01

    New nonimmortalized fibroblast-like cell lines SC5-MSC and SC3a-MSC, FetMSC, FRSN were obtained from human embryonic stem cells (ESC), bone marrow of a 5-6-days embryo and foreskin of a 3-years-old boy, respectively. All the lines are successfully used as the feeder at human ESC cultivation. It is determined that the average cell population doublings time varies from 25.5 h for ISC5-MSC to 38.8 h for SC3a-MSC. Active proliferation of all the lines is also shown by the corresponding growth curves. Numerical and structural karyotypic analysis showed that these lines had normal karyotype: 46,XX (SC5-MSC and SC3a-MSC) and 46,XY (FetMSC and FRSN). To determine the status of the lines, their cell surface markers were analyzed by flow cytometry. This analysis revealed the presence of surface antigens CD44, CD73, CD90, CD105 and HLA-ABC, characteristic of human MSC, and the absence of CD34 and HLA-DR. Different lines were found to express CD117(c-kit) to a different level. Immunofluorescence and flow cytometry analysis did not detect TRA-1-60 and Oct-4, characteristic of human embryonic stem cells, and revealed interlinear variations in the level of SSEA, which did not depend on the cell origin. It is not clear yet whether these interlinear variations affect functional MSC status. In all the lines, immunofluorescence analysis showed the presence of the markers of early differentiation in the derivates of three germ layers which may allow MSC to be useful, in corresponding microenvironments, for reparation of tissue injures. Adipogenic and osteogenic differentiatiation of all cell lines has been shown.

  1. Novel cancer-testis antigen expression on glioma cell lines derived from high-grade glioma patients.

    PubMed

    Akiyama, Yasuto; Komiyama, Masaru; Miyata, Haruo; Yagoto, Mika; Ashizawa, Tadashi; Iizuka, Akira; Oshita, Chie; Kume, Akiko; Nogami, Masahiro; Ito, Ichiro; Watanabe, Reiko; Sugino, Takashi; Mitsuya, Koichi; Hayashi, Nakamasa; Nakasu, Yoko; Yamaguchi, Ken

    2014-04-01

    Glioblastoma multiforme (GBM) is one of the most malignant and aggressive tumors, and has a very poor prognosis with a mean survival time of <2 years, despite intensive treatment using chemo-radiation. Therefore, novel therapeutic approaches including immunotherapy have been developed against GBM. For the purpose of identifying novel target antigens contributing to GBM treatment, we developed 17 primary glioma cell lines derived from high-grade glioma patients, and analyzed the expression of various tumor antigens and glioma-associated markers using a quantitative PCR and immunohistochemistry (IHC). A quantitative PCR using 54 cancer-testis (CT) antigen-specific primers showed that 36 CT antigens were positive in at least 1 of 17 serum-derived cell lines, and 17 antigens were positive in >50% cell lines. Impressively, 6 genes (BAGE, MAGE-A12, CASC5, CTAGE1, DDX43 and IL-13RA2) were detected in all cell lines. The expression of other 13 glioma-associated antigens than CT genes were also investigated, and 10 genes were detected in >70% cell lines. The expression of CT antigen and glioma-associated antigen genes with a high frequency were also verified in IHC analysis. Moreover, a relationship of antigen gene expressions with a high frequency to overall survival was investigated using the Repository of Molecular Brain Neoplasia Data (REMBRANDT) database of the National Cancer Institute, and expression of 6 genes including IL-13RA2 was inversely correlated to overall survival time. Furthermore, 4 genes including DDX43, TDRD1, HER2 and gp100 were identified as MGMT-relevant factors. In the present study, several CT antigen including novel genes were detected in high-grade glioma primary cell lines, which might contribute to developing novel immunotherapy and glioma-specific biomarkers in future.

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

    PubMed

    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.

  3. Characterization of forebrain neurons derived from late-onset Huntington's disease human embryonic stem cell lines

    PubMed Central

    Niclis, Jonathan C.; Pinar, Anita; Haynes, John M.; Alsanie, Walaa; Jenny, Robert; Dottori, Mirella; Cram, David S.

    2012-01-01

    Huntington's disease (HD) is an incurable neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the Huntingtin (HTT) gene. Recently, induced pluripotent stem cell (iPSC) lines carrying atypical and aggressive (CAG60+) HD variants have been generated and exhibit disparate molecular pathologies. Here we investigate two human embryonic stem cell (hESC) lines carrying CAG37 and CAG51 typical late-onset repeat expansions in comparison to wildtype control lines during 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-evoked responses were observed in HD CAG51 neurons. These findings suggest typical late-onset HD mutations do not alter pluripotent parameters or the capacity to generate forebrain neurons, but that such progeny may recapitulate hallmarks observed in established HD model systems. 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. PMID:23576953

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

    PubMed

    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.

  5. Temperature-dependent growth rates and gene expression patterns of various medaka Oryzias latipes cell lines derived from different populations.

    PubMed

    Hirayama, Makoto; Mitani, Hiroshi; Watabe, Shugo

    2006-05-01

    Medaka Oryzias latipes has several geographically and genetically distinct populations. We examined temperature acclimation response in various medaka cell lines derived from different populations. Measurement of cell growth at various temperatures suggested that 15 degrees Celsius was the permissive growth temperature in all cell lines from the Northern Japanese and East Korean populations, but not in those from the Southern Japanese population and medaka-related species Oryzias celebensis, which inhabits a tropical zone. RT-PCR for 102 temperature-responsive genes, previously reported in other species, revealed that the accumulated mRNA level of a gene encoding HSP47 was lower at 25 degrees Celsius than at 33 degrees Celsius, and vice versa for 12 genes including IkappaBalpha and Rab-1c, in OLHNI-1 cell line from the Northern Japanese population. Further analysis by real-time PCR demonstrated that the accumulated mRNA levels of IkappaBalpha and Rab-1c in OLHNI-1 and OLSOK-e7 cell lines from the East Korean population were increased when the culture temperature was shifted from 33 to 15 degrees Celsius, but not in OLHdrR-e3 cell line from the Southern Japanese population. Since IkappaBalpha and Rab-1c are related to the NFkappaB cascade and endoplasmic reticulum-to-Golgi transport, respectively, it is inferred that immune responses and intracellular transport are possibly critical to temperature adaptation for medaka.

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

    PubMed

    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.

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

    PubMed

    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.

  8. A general solution to the hidden-line problem. [to graphically represent aerodynamic stability derivatives

    NASA Technical Reports Server (NTRS)

    Hedgley, D. R., Jr.

    1982-01-01

    The requirements for computer-generated perspective projections of three dimensional objects has escalated. A general solution was developed. The theoretical solution to this problem is presented. The method is very efficient as it minimizes the selection of points and comparison of line segments and hence avoids the devastation of square-law growth.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

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

    PubMed

    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.

  11. Characterization of Murine Pituitary-Derived Cell Lines Tpit/F1, Tpit/E and TtT/GF

    PubMed Central

    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. PMID:24881870

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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

  14. Use of SSR markers to determine the anther-derived homozygous lines in coconut.

    PubMed

    Perera, P I P; Perera, L; Hocher, V; Verdeil, J-L; Yakandawala, D M D; Weerakoon, L K

    2008-11-01

    Anther culture was used to obtain dihaploid (DH) coconut plants and their ploidy level was determined by flow cytometric analysis. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. Ploidy analysis showed that 50% of the tested plantlets were haploid and 50% were diploid. Polymorphic fragments of the mother palm and their segregation patterns in anther-derived plantlets were used to determine the origin of the diploid plantlets. Using a diagnostic SSR marker (CNZ43), all the diploid plantlets tested were identified as being derived from microspores (i.e. were homozygous) and were thus candidates for use in coconut breeding programs.

  15. Identification and characterisation in vitro of cells with a non-SCLC cell-like phenotype derived from a continuous SCLC cell line.

    PubMed

    Khan, M Z; Freshney, R I; Murray, A M; Merry, S; Plumb, J A; McNicol, A M

    1991-01-01

    Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth rates of both the derivative lines were faster than the parental line with doubling times closer to non-SCLC cell lines in the derivative lines. Both H69V and H69VZ either express very low levels or do not express neuroendocrine cell markers including L-dopa-decarboxylase (DDC), creatine kinase-BB isoenzyme (CK-BB), bombesin-like immunoreactivity (BLI), neuron specific enolase (NSE), and neurosecretory type dense core granules (DGCs), compared to the parental cell line. All the lines stained positive for epithelial markers such as CAM5.2. LDH isoenzyme and chromosome analyses confirmed the human origin of all the cell lines. Therefore, it appears that cell line NCI-H69 contains stem cell subpopulation capable of generating cells of both small and non-small cell like phenotypes.

  16. Oxygen abundances derived from UV OH and O I IR lines in very metal-poor stars

    NASA Astrophysics Data System (ADS)

    García López, Ramón J.; Israelian, Garik; Rebolo, Rafael; Bonifacio, Piercarlo; Molaro, Paolo; Basri, Gibor; Shchukina, Natalya

    Oxygen abundances have been derived in a sample of very metal-poor stars using the O I triplet at λλ7771-5 Å and OH lines in the near UV. A detailed NLTE analysis of iron lines has been carried out for one of the observed stars, BD +23°3130, providing consistent values of effective temperature and surface gravity that are in very good agreement with independent estimates from the infrared flux method and Hipparcos parallaxes, respectively. These parameters, especially the higher gravity obtained with respect to previous analyses, reduce the discrepancies claimed between the oxygen abundances determined from OH, O I triplet and [O I] λ6300 Å lines, and give consistent abundances to within 0.16 dex for BD +23°3130 ([Fe/H]NLTE = -2.43). The oxygen abundances derived for this new sample confirm previous findings for a progressive linear increase in the oxygen-to-iron ratio with a slope -0.33±0.02 (including NLTE corrections to the iron abundances for all the stars considered) from solar metallicity to [Fe/H]~ -3, and [O/Fe] values as high as ~1.1 for stars with [Fe/H]<~ -2.5. These results can be interpreted as evidence for oxygen overproduction in the very early epoch of the formation of the Galactic halo, possibly associated with supernova events with very massive progenitor stars.

  17. Chromosome elimination and in vivo haploid production induced by Stock 6-derived inducer line in maize (Zea mays L.).

    PubMed

    Zhang, Zili; Qiu, Fazhan; Liu, Yongzhong; Ma, Kejun; Li, Zaiyun; Xu, Shangzhong

    2008-12-01

    In vivo haploid production induced by inducer lines derived from Stock 6 is widely used in breeding program of maize (Zea mays L.), but the mechanisms behind have not yet been fully understood. In this study, average frequency of haploid induction in four inbred lines by Stock 6-derived inducer line HZI1 was above 10%. About 0.2% kernels from the cross Hua24 x HZI1 had mosaic endosperm showing yellow shrunken parts from Hua24 to normal parts with purple aleurone from HZI1. Individual lagged chromosomes and micronuclei were observed in mitotic cells of ovules pollinated by HZI1. Above 56.4% of the radicles from the kernels with purple aleurone and colorless embryos were mixoploid (2n = 9-21), and more than 45.22% cells were haploid cells (2n = 10) in three crosses. More than 62.5% of the radicles from the kernels with purple aleurone and purple embryos were mixoploid (2n = 9-21) having 54.27% cells with 2n = 20. SSR analysis showed that all haploids from the cross Hua24 x HZI1 shared the same genomic compositions as Hua24 except for plants Nos. 862 and 857 with some polymorphic DNA bands. The results revealed that chromosome elimination after fertilization caused the haploid production in maize.

  18. The genome landscape of the african green monkey kidney-derived vero cell line.

    PubMed

    Osada, Naoki; Kohara, Arihiro; Yamaji, Toshiyuki; Hirayama, Noriko; Kasai, Fumio; Sekizuka, Tsuyoshi; Kuroda, Makoto; Hanada, Kentaro

    2014-12-01

    Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells.

  19. Thyrotropin dependent and independent thyroid cell lines selected from FRTL-5 derived tumors grown in nude mice

    SciTech Connect

    Ossendorp, F.A.; Bruning, P.F.; Schuuring, E.M.; Van Den Brink, J.A.; van der Heide, D.; De Vijlder, J.J.; De Bruin, T.W. )

    1990-07-01

    FRTL-5 cells were used to set up a thyroid tumor model system in C3H nu/nu mice. FRTL-5 tumors could be grown in nude mice provided serum TSH levels were elevated. Persistent TSH elevation was obtained by administration of Na131I, rendering the mice hypothyroid. After 4 weeks FRTL-5 cells were injected sc resulting in tumor growth within 2 weeks in eight out of eight mice. Although the tumors showed an apparently undifferentiated histology, lacking normal follicular structures, they were functional since the tumors were capable of concentrating (131)iodine, as demonstrated by nuclear imaging. From one of the tumors a new cell line was isolated (FRTL-5/T) that, like the parental FRTL-5 cell line, was TSH dependent for growth. In a control group of six euthyroid nude mice FRTL-5 tumor growth could not be obtained with one exception. After 3 months one animal developed a small tumor that grew rapidly thereafter. This tumor was easily transplantable in other euthyroid nude mice, showed an undifferentiated histology, and was nonfunctional, as it could not concentrate (131)iodine. From this tumor two cell lines were derived: one cultured in the presence of TSH (FRTL-5/TP) and one in the absence of TSH (FRTL-5/TA). The cell lines were analyzed for TSH responsive functions and TSH receptor expression. Responsiveness to TSH in FRTL-5/T and the parental FRTL-5 cell line were similar for most thyroid specific functions tested. However, FRTL-5/T was less sensitive than FRTL-5 for TSH induced (3H)thymidine incorporation. Both cell lines had two classes of TSH binding sites with high and low affinity respectively. FRTL-5/TP and FRTL-5/TA were both able to grow in TSH free medium and were nonresponsive to TSH in vitro, as tested for (3H)thymidine and (3H)uridine incorporation, iodine uptake, thyroglobulin iodination, and thyroglobulin secretion.

  20. Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

    PubMed

    Gleich, Alexander; Kaiser, Bastian; Schumann, Julia; Fuhrmann, Herbert

    2016-06-01

    Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 μM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

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

    PubMed

    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.

  2. Antimicrobial resistance (AMR) and plant-derived antimicrobials (PDAms) as an alternative drug line to control infections.

    PubMed

    Srivastava, Jatin; Chandra, Harish; Nautiyal, Anant R; Kalra, Swinder J S

    2014-10-01

    Infectious diseases caused by antimicrobial-resistant microbes (ARMs) and the treatment are the serious problems in the field of medical science today world over. The development of alternative drug line to treat such infectious diseases is urgently required. Researches on ARMs revealed the presence of membrane proteins responsible for effusing the antibiotics from the bacterial cells. Such proteins have successfully been treated by plant-derived antimicrobials (PDAms) synergistically along with the commercially available antibiotics. Such synergistic action usually inhibits the efflux pump. The enhanced activity of plant-derived antimicrobials is being researched and is considered as the future treatment strategy to cure the incurable infections. The present paper reviews the advancement made in the researches on antimicrobial resistance along with the discovery and the development of more active PDAms.

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

    PubMed

    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.

  4. Characterization of a myoepithelial cell line derived from a neonatal rat mammary gland

    PubMed Central

    1981-01-01

    A clonal, myoepithelial-like cell line has been obtained from a primary culture established from the mammary gland of a 7-d-old rat. In a number of respects, this cell line, termed Rama 401, resembles the myoepithelial cells of the mammary gland, especially when grown on floating collagen gels. The cells grow as multilayers on the gel surface and form branching structures that do not appear to contain a lumen. They are rather elongated, with irregular-shaped, flattened nuclei that contain large amounts of peripheral chromatin. Elongated processes project from the cell surface and numerous membrane pinocytotic vesicles can be seen. The cytoplasm is filled with linear arrays of 5- to 7-nm filaments with occasional dense foci. Cell junctions with associated 8- to 11-nm tonofilaments are also observed. Immunofluorescence techniques reveal actin and myosin filaments and also intermediate filaments of both prekeratin and vimentin types. Rama 401 cells secrete an amorphous material that, when an immunoperoxidase technique is used, stains with antibodies to basement membrane-specific type IV collagen. Localized densities of the cell membrane, which resemble hemidesmosomes, are located adjacent to these extracellular deposits. Immunofluorescence staining and immunoprecipitation techniques reveal that the cells also synthesize two other basement membrane proteins, laminin and fibronectin. The type IV collagen consists of two chains with molecular weights of 195,000 and 185,000. PMID:7199047

  5. Germline transmission of an embryonic stem cell line derived from BALB/c cataract mice.

    PubMed

    Peng, Xinrong; Liu, Tao; Shi, Chuanyin; Zhang, Liqing; Wang, Ying; Zhao, Wuyang; Jiang, Lihua; Wu, Mengchao; Zhang, Yong; Qian, Qijun

    2014-01-01

    Mice embryonic stem (ES) cells have enabled the generation of mouse strains with defined mutation(s) in their genome for putative disease loci analysis. In the study of cataract, the complex genetic background of this disease and lack of long-term self-renewal ES cells have hampered the functional researches of cataract-related genes. In this study, we aimed to establish ES cells from inherited cataract mice (BALB/CCat/Cat). Embryos of cataract mice were cultured in chemical-defined N2B27 medium with the presence of two small molecules PD0325901 and CHIR99021 (2i) and an ES cell line (named EH-BES) was successfully established. EH-BES showed long-term self-renewal in 2i medium and maintained capacity of germline transmission. Most importantly, the produced chimera and offspring developed congenital cataract as well. Flow cytometry assay revealed that EH-BES are homogeneous in expression of Oct4 and Rex1in 2i medium, which may account for their self-renewal ability. With long-term self-renewal ability and germline-competent, EH-BES cell line can facilitate genetic and functional researches of cataract-related genes and better address mechanisms of cataract.

  6. 3-Methyl pyruvate enhances radiosensitivity through increasing mitochondria-derived reactive oxygen species in tumor cell lines.

    PubMed

    Nishida, Naoya; Yasui, Hironobu; Nagane, Masaki; Yamamori, Tohru; Inanami, Osamu

    2014-05-01

    Considerable interest has recently been focused on the special characteristics of cancer metabolism, and several drugs designed to modulate cancer metabolism have been tested as potential anticancer agents. To date, however, very few studies have been conducted to investigate the combined effects of anticancer drugs and radiotherapy. In this study, to evaluate the role of mitochondria-derived reactive oxygen species (ROS) in the radiation-induced cell death of tumor cells, we have examined the effect of 3-methyl pyruvate (MP). MP is a membrane-permeable pyruvate derivative that is capable of activating mitochondrial energy metabolism in human lung carcinoma A549 cells and murine squamous carcinoma SCCVII cells. Pretreatment with MP significantly enhanced radiation-induced cell death in both cell lines, and also led to increases in the mitochondrial membrane potential, intracellular adenosine triphosphate content, and mitochondria-derived ROS production following the exposure of the cells to X-rays. In A549 cells, MP-induced radiosensitization was completely abolished by vitamin C. In contrast, it was partially abolished in SCCVII cells. These results therefore suggest that the treatment of the cells with MP induced radiosensitization via the production of excess mitochondria-derived ROS in tumor cells.

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

    SciTech Connect

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

    2009-05-15

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

  8. Derivation of transplantable 7,12-dimethylbenz(a)anthracene-induced chicken fibrosarcoma lines: differences in metastasizing properties and organ specificity

    SciTech Connect

    Galton, J.E.; Xue, B.; Hochwald, G.M.; Thorbecke, G.J.

    1982-08-01

    Transplantable 7,12-dimethylbenz(a)anthracene-induced SC chicken fibrosarcoma (CHCT-NYU) lines were studied for their ability to grow in internal organs after iv injection (artificial metastases) into 1- to 3-week-old chickens. Some tumor lines were recently derived, whereas others were studied after many serial subcutaneous transplantations. Artificial metastases were seen in the stomach, pancreas, lungs, heart, and muscle, and occasionally in the kidneys and liver. Agammaglobulinemic recipients showed more extensive organ involvement than normal recipients of the same age. Whole-body ..gamma..-irradiation enhanced the incidence of artificial metastases, particularly in lungs. Antibody from the serum of a primary tumor-bearing host reduced the growth of the corresponding tumor in many organs. The metastatic pattern of line CHCT-NYU4 was a relatively stable property. However, intravenous transplantation of tumor cells from line CHCT-NYU4 taken from the liver, lungs, and pancreas of a single recipient established sublines with changes in organ specificity. After a few such serial transplants of liver-derived tumor, a line was derived that grew virtually in the liver alone. A subline with preference for growth in lungs was also obtained, but its ability to grow in the pancreas persisted. A pancreas-derived tumor line also grew in the liver and lungs. Subcutaneous transplants of tissue fragments of the lung-derived tumor line caused the appearance of spontaneous metastases in lungs. The incidence of spontaneous metastases with the lung-derived line was much greater than that with the liver-derived line or with the original CHCT-NYU4 line.

  9. Multichannel spectroreflectometry: a noninvasive method for assessment of on-line hemoglobin derivatives.

    PubMed

    Diaconu, Vasile

    2009-04-01

    The goal of the current study was to introduce a mathematical method to derive hemoglobin, oxyhemoglobin and carboxyl-hemoglobin absorption factors from full spectrum reflectometry measurements of retinal microcapillaries. The mathematical equation that describes the spectral reflectometry function was expressed as a linear combination of several terms of S(i)(lambda) representing the spectral signature functions of hemoglobin, oxyhemoglobin, carboxyl-hemoglobin, ocular media, melanin, and a scattering factor. Contrary to the classical model, where the reflectometry function was expressed as an absorbance Ab(lambda)=log?(incident light(lambda)/reflected light(lambda)), in this model and system, it is proposed to express the reflectometry function from the eye structures as an absorption factor A(lambda)%=incident light(lambda)/reflected light(lambda). To increase confidence in the estimation of hemoglobin derivatives, the mathematical model was applied to only a part of the spectral function of reflectometry, while the results of the model were used to explain the other part of the reflectometry function. The results demonstrate that for the visible spectral field, the model that explains the absorption of the light by the blood contained in the microcapillaries of biological structures is not compatible with the Beer-Lambert law.

  10. Effects of brain-derived and glial cell line-derived neurotrophic factors on startle response and disrupted prepulse inhibition in mice of DBA/2J inbred strain.

    PubMed

    Naumenko, Vladimir S; Bazovkina, Daria V; Morozova, Maryana V; Popova, Nina K

    2013-08-29

    Prepulse inhibition (PPI), the reduction in acoustic startle reflex when it is preceded by weak prepulse stimuli, is a measure of critical to normal brain functioning sensorimotor gating. PPI deficit was shown in a variety of psychiatric disorders including schizophrenia, and in DBA/2J mouse strain. In the current study, we examined the effects of brain-derived (BDNF) and glial cell line-derived (GDNF) neurotrophic factors on acoustic startle response and PPI in DBA/2J mice. It was found that BDNF (300 ng, i.c.v.) significantly increased amplitude of startle response and restored disrupted PPI in 7 days after acute administration. GDNF (800 ng, i.c.v.) did not produce significant alteration neither in amplitude of startle response nor in PPI in DBA/2J mice. The reversal effect of BDNF on PPI deficit was unusually long-lasting: significant increase in PPI was found 1.5 months after single acute BDNF administration. Long-term ameliorative effect BDNF on disrupted PPI suggested the implication of epigenetic mechanism in BDNF action on neurogenesis. BDNF rather than GDNF could be a perspective drug for the treatment of sensorimotor gating impairments.

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

    PubMed

    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.

  12. PAX8 is transcribed aberrantly in cervical tumors and derived cell lines due to complex gene rearrangements.

    PubMed

    López-Urrutia, Eduardo; Pedroza-Torres, Abraham; Fernández-Retana, Jorge; De Leon, David Cantu; Morales-González, Fermín; Jacobo-Herrera, Nadia; Peralta-Zaragoza, Oscar; García-Mendez, Jorge; García-Castillo, Verónica; Bautista-Isidro, Osvaldo; Pérez-Plasencia, Carlos

    2016-07-01

    The transcription factor PAX8, a member of the paired box-containing gene family with an important role in embryogenesis of the kidney, thyroid gland and nervous system, has been described as a biomarker in tumors of the thyroid, parathyroid, kidney and thymus. The PAX8 gene gives rise to four isoforms, through alternative mRNA splicing, but the splicing pattern in tumors is not yet established. Cervical cancer has a positive expression of PAX8; however, there is no available data determining which PAX8 isoform or isoforms are present in cervical cancer tissues as well as in cervical carcinoma-derived cell lines. Instead of a differential pattern of splicing isoforms, we found numerous previously unreported PAX8 aberrant transcripts ranging from 378 to 542 bases and present in both cervical carcinoma-derived cell lines and tumor samples. This is the first report of PAX8 aberrant transcript production in cervical cancer. Reported PAX8 isoforms possess differential transactivation properties; therefore, besides being a helpful marker for detection of cancer, PAX8 isoforms can plausibly exert differential regulation properties during carcinogenesis.

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

    NASA Astrophysics Data System (ADS)

    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.

  14. On-line NMR detection of microgram quantities of heparin-derived oligosaccharides and their structure elucidation by microcoil NMR.

    PubMed

    Korir, Albert K; Larive, Cynthia K

    2007-08-01

    The isolation and purification of sufficient quantities of heparin-derived oligosaccharides for characterization by NMR is a tedious and time-consuming process. In addition, the structural complexity and microheterogeneity of heparin makes its characterization a challenging task. The improved mass-sensitivity of microcoil NMR probe technology makes this technique well suited for characterization of mass-limited heparin-derived oligosaccharides. Although microcoil probes have poorer concentration sensitivity than conventional NMR probes, this limitation can be overcome by coupling capillary isotachophoresis (cITP) with on-line microcoil NMR detection (cITP-NMR). Strategies to improve the sensitivity of on-line NMR detection through changes in probe design and in the cITP-NMR experimental protocol are discussed. These improvements in sensitivity allow acquisition of cITP-NMR survey spectra facilitating tentative identification of unknown oligosaccharides. Complete structure elucidation for microgram quantities of the purified material can be carried out through acquisition of 2D NMR spectra using a CapNMR microcoil probe.

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

    PubMed

    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 < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated 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 < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These 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

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    Chauhan, Vinita; Howland, Matthew; Wilkins, Ruth

    2012-01-01

    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. PMID:22619631

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

    PubMed Central

    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

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

    PubMed Central

    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

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

    PubMed Central

    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

  2. Accelerated Senescence and Enhanced Disease Resistance in Hybrid Chlorosis Lines Derived from Interspecific Crosses between Tetraploid Wheat and Aegilops tauschii

    PubMed Central

    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. PMID:25806790

  3. A comparitive assessement of cytokine expression in human-derived cell lines exposed to alpha particles and X-rays.

    PubMed

    Chauhan, Vinita; Howland, Matthew; Wilkins, Ruth

    2012-01-01

    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.

  4. Adaptive responses to osmotic stress in kidney-derived cell lines from Scatophagus argus, a euryhaline fish.

    PubMed

    Gui, Lang; Zhang, Peipei; Liang, Xuemei; Su, Maoliang; Wu, Di; Zhang, Junbin

    2016-06-01

    The euryhaline fish, the spotted scat (Scatophagus argus), is exceptional for its ability to tolerate rapid fluctuations in salinity. To better understand fish osmoregulation and enable more precise analyses of specific features of adaptive responses to the osmotic stress in fish, a S. argus kidney-derived cell line (SK) was developed and subcultured for more than 70 passages. The cells were mostly fibroblast-like, with a normal diploid karyotype (2n=48). A low-osmolarity-adapted SK cell line (SK-la) was induced by growth in a hypotonic solution (150 mOsm). Effects of different osmotic stresses (150, 300 and 450 mOsm) on cell growth, cell morphology, cell volume changes and cell damage in SK, SK-la and CIK (a kidney-derived cell line from freshwater grass carp) cells were studied. These were compared by use of microscopic observation, flow cytometry and a Na-K-ATPase (NKA) assay. SK cells became smaller and grew rapidly in response to hypotonic stress (150 mOsm), and exhibited no visible morphological changes in response to hypertonic stress (450 mOsm). SK-la grew well by moderate hypertonicity (300 mOsm) but depressed in severe hypertonicity (450 mOsm), the number of unhealthy SK-la cells rose as osmolarity increased. In contrast, CIK cells became unhealthy with anisotonic challenge. The NKA activities of SK and CIK cells were assayed after exposure to anisotonic conditions, and rapid decreases were detected immediately except SK cells which were not affected in hypotonicity. Unlike in SK and CIK, an increase following a down-regulation of NKA activity was observed in SK-la cells upon moderate hypertonic stress. These results suggested that SK and SK-la cells had stronger osmoregulatory capacity than CIK cells, and provided new insights on the osmosensing and osmotic adaption in euryhaline fish kidney.

  5. An approach to derive regional snow lines and glacier mass change from MODIS imagery, western North America

    NASA Astrophysics Data System (ADS)

    Shea, J. M.; Menounos, B.; Moore, R. D.; Tennant, C.

    2013-04-01

    We describe a method to calculate regional snow line elevations and annual equilibrium line altitudes (ELAs) from daily MODIS imagery (MOD02QKM) on large glaciers and icefields in western North America. An automated cluster analysis of the cloud-masked visible and near-infrared bands at 250 m resolution is used to delineate glacier facies (snow and ice) for ten glacierized regions between 2000-2011. For each region and season, the maximum observed value of the 20th percentile of snow-covered pixels (ZS(20)) is used to define a regional ELA proxy (ELAest). Our results indicate significant increases in the regional ELA proxy at two continental sites (Peyto Glacier and Gulkana Glacier) over the period of observation, though no statistically significant trends are identified at other sites. To evaluate the utility of regional ELA proxies derived from MOD02QKM imagery, we compare standard geodetic estimates of glacier mass change with estimates derived from historical mass balance gradients and observations of ZS(20) at three large icefields. Our approach yields estimates of mass change that more negative than traditional geodetic approaches, though MODIS-derived estimates are within the margins of error at all three sites. Both estimates of glacier mass change corroborate the continued mass loss of glaciers in western North America. Between 2000 and 2009, the geodetic change approach yields mean annual rates of surface elevation change for the Columbia, Lillooet, and Sittakanay icefields of -0.29 ± 0.05, -0.26 ± 0.05, and -0.63 ± 0.17 m a-1, respectively. This study provides a new technique for glacier facies detection at daily timescales, and contributes to the development of regional estimates of glacier mass change, both of which are critical for studies of glacier contributions to streamflow and global sea level rise.

  6. Epstein-Barr virus genetic variation in lymphoblastoid cell lines derived from Kenyan pediatric population.

    PubMed

    Simbiri, Kenneth O; Smith, Nicholas A; Otieno, Richard; Wohlford, Eric E M; Daud, Ibrahim I; Odada, Sumba P; Middleton, Frank; Rochford, Rosemary

    2015-01-01

    Epstein-Barr virus (EBV) is associated with Burkitt's lymphoma (BL), and in regions of sub-Saharan Africa where endemic BL is common, both the EBV Type 1 (EBV-1) and EBV Type 2 strains (EBV-2) are found. Little is known about genetic variation of EBV strains in areas of sub-Saharan Africa. In the present study, spontaneous lymphoblastoid cell lines (LCLs) were generated from samples obtained from Kenya. Polymerase chain reaction (PCR) amplification of the EBV genome was done using multiple primers and sequenced by next-generation sequencing (NGS). Phylogenetic analyses against the published EBV-1 and EBV-2 strains indicated that one sample, LCL10 was closely related to EBV-2, while the remaining 3 LCL samples were more closely related to EBV-1. Moreover, single nucleotide polymorphism (SNP) analyses showed clustering of LCL variants. We further show by analysis of EBNA-1, BLLF1, BPLF1, and BRRF2 that latent genes are less conserved than lytic genes in these LCLs from a single geographic region. In this study we have shown that NGS is highly useful for deciphering detailed inter and intra-variations in EBV genomes and that within a geographic region different EBV genetic variations can co-exist, the implications of which warrant further investigation. The findings will enhance our understanding of potential pathogenic variants critical to the development and maintenance of EBV-associated malignancies.

  7. Genomic deletions in cell lines derived from primitive neuroectodermal tumors of the central nervous system.

    PubMed

    Dallas, Peter B; Terry, Philippa A; Kees, Ursula R

    2005-06-01

    Extensive genomic deletions affecting a variety of chromosomes are a common finding in primitive neuroectodermal tumors of the central nervous system (CNS-PNETs), implicating the loss of multiple tumor suppressor genes in the pathogenesis of these tumors. We have used representational difference analysis, microsatellite mapping, and quantitative polymerase chain reaction to identify and verify the presence of genomic deletions on a number of chromosomes in CNS-PNET cell lines. This systematic approach has confirmed the importance of deletions at 10q, 16q, and 17p in PNET pathology and has revealed other regions of deletion not commonly described (e.g., Xq, 1p, 7p, and 13q). These data highlight the prevalence of hemizygous loss in CNS-PNET cells, suggesting that haploinsufficiency affecting multiple tumor suppressor genes may play a fundamental role in CNS-PNET pathogenesis. The identification of specific genes and signaling pathways that are compromised in CNS-PNET cells is crucial for development of more efficacious and less invasive treatments, as are urgently needed.

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

    PubMed

    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.

  9. Parental genome contribution in maize DH lines derived from six backcross populations using genotyping by sequencing and its relationship with testcross performance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular characterization of doubled haploid (DH) maize lines and estimation of parental genome contribution (PGC) may be useful for choosing pairs of DH lines for hybrid make up and new pedigree starts. Six BC1-derived DH populations created by crossing two donor with three recurrent parents were ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  11. Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line

    SciTech Connect

    Blusztajn, J.K.; Liscovitch, M.; Richardson, U.I.

    1987-08-01

    Cholinergic neurons are unique among cells since they alone utilize choline not only as a component of major membrane phospholipids, such as phosphatidylcholine (Ptd-Cho), but also as a precursor of their neurotransmitter acetylcholine (AcCho). It has been hypothesized that choline-phospholipids might serve as a storage pool of choline for AcCho synthesis. The selective vulnerability of cholinergic neurons in certain neurodegenerative diseases (e.g., Alzheimer disease, motor neuron disorders) might result from the abnormally accelerated liberation of choline (to be used a precursor of AcCho) from membrane phospholipids, resulting in altered membrane composition and function and compromised neuronal viability. However, the proposed metabolic link between membrane turnover and AcCho synthesis has been difficult to demonstrate because of the heterogeneity of the preparations used. Here the authors used a population of purely cholinergic cells (human neuroblastomas, LA-N-2), incubated in the presence of (methyl-/sup 3/H)methionine to selectively label PtdCho synthesized by methylation of phosphatidylethanolamine, the only pathway of de novo choline synthesis. Three peaks of radioactive material that cochromatographed with authentic AcCho, choline, and phosphocholine were observed when the water-soluble metabolites of the (/sup 3/H)PtdCho were purified by high-performance liquid chromatography. The results demonstrate that AcCho can be synthesized from choline derived from the degradation of endogenous PtdCho formed de novo by methylation of phosphatidylethanolamine.

  12. Tumor suppression by MEG3 lncRNA in a human pituitary tumor derived cell line.

    PubMed

    Chunharojrith, Paweena; Nakayama, Yuki; Jiang, Xiaobing; Kery, Rachel E; Ma, Jun; De La Hoz Ulloa, Cristine S; Zhang, Xun; Zhou, Yunli; Klibanski, Anne

    2015-11-15

    Human clinically non-functioning pituitary adenomas (NFAs) account for approximately 40% of diagnosed pituitary tumors. Epigenetic mutations in tumor suppressive genes play an important role in NFA development. Maternally expressed gene 3 (MEG3) is a long non-coding RNA (lncRNA) and we hypothesized that it is a candidate tumor suppressor whose epigenetic silencing is specifically linked to NFA development. In this study, we introduced MEG3 expression into PDFS cells, derived from a human NFA, using both inducible and constitutively active expression systems. MEG3 expression significantly suppressed xenograft tumor growth in vivo in nude mice. When induced in culture, MEG3 caused cell cycle arrest at the G1 phase. In addition, inactivation of p53 completely abolished tumor suppression by MEG3, indicating that MEG3 tumor suppression is mediated by p53. In conclusion, our data support the hypothesis that MEG3 is a lncRNA tumor suppressor in the pituitary and its inactivation contributes to NFA development.

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

    PubMed

    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. Apical uptake of choline and cationic drugs in epithelial cell lines derived from human placenta.

    PubMed

    Müller, J; Born, I; Neubert, R H; Brandsch, M

    2005-01-01

    Many cationic drugs are administered during pregnancy and might enter the fetal circulation by transplacental passage. This study was performed to characterize the apical uptake of choline and several cationic drugs at cultured epithelial cells of the human placenta. Total uptake of [3H]choline in BeWo cells was H(+)-independent and to 65% Na(+)-independent. Uptake rates into both cell lines were saturable with Michaelis-Menten constants (Kt) of 108 microM (BeWo) and 206 microM (JEG-3), respectively. Cationic drugs such as etilefrine, clonidine, ranitidine, diphenhydramine, imipramine and butylscopolamine strongly inhibited the [3H]choline uptake in BeWo cells and in JEG-3 cells, with Ki values ranging from 0.18 to 3.3 mM. In contrast, tetraethylammonium had only little inhibitory effect on [3H]choline uptake. Using high-performance capillary electrophoresis for quantitative analyses, uptake of etilefrine and diphenhydramine into JEG-3 or BeWo cells was measured. Diphenhydramine was transported into JEG-3 cells in a saturable manner with a Kt value of 0.75 mM. In the presence of sodium, diphenhydramine uptake at BeWo cells was inhibited to 69% by the addition of 50 mM choline chloride. Like choline uptake, total diphenhydramine uptake was to 68% Na(+)-independent in BeWo cells. We conclude that in addition to choline, several cationic drugs, in particular diphenhydramine, are taken up by placental epithelial cells from the maternal blood by carrier-mediated processes. Etilefrine, clonidine, ranitidine, diphenhydramine and butylscopolamine interact with the Na(+)-independent placental choline transport system.

  15. Comparative oncological studies of feline bronchioloalveolar lung carcinoma, its derived cell line and xenograft.

    PubMed

    Grossman, Deborah A; Hiti, Alan L; McNiel, Elizabeth A; Ye, Yin; Alpaugh, Mary L; Barsky, Sanford H

    2002-07-01

    Although certain neoplasms are unique to man, others occur across species. One such neoplasm is bronchioloalveolar lung carcinoma (BAC), a neoplasm of the Type II pneumocyte that affects humans, sheep, and small animals (dogs and cats). Human BAC occurs largely in nonsmokers. Sheep BAC is caused by the jaagsiekte retrovirus and is endemic and contagious. Feline BAC is neither endemic nor contagious and occurs sporadically and spontaneously in older purebred cats. In these respects, feline BAC is more closely similar to human BAC than sheep BAC (jaagsiekte) is. To study feline BAC further, we established the first immortal cell line (SPARKY) and transplantable scid mouse xenograft (Sparky-X) from a malignant pleural effusion of a 12-year-old Persian male with autopsy-confirmed BAC. SPARKY exhibited a Type II pneumocyte phenotype characterized by surfactant and thyroid-transcription factor-1 immunoreactivities and lamellar bodies. SPARKY's karyotype was aneuploid (66 chromosomes: 38, normal cat) and showed evidence of genomic instability analogous to human lung cancers. p53 showed a homozygous G to T transversion at codon 167, the feline equivalent of human codon 175, one of the many hot spots mutated in the lung cancers of smokers. H-ras and K-ras were not altered. By reverse transcription-PCR, SPARKY lacked expression of retroviral JSRVgag transcripts that were present in the lungs of sheep BAC (jaagsiekte). Unlike human BAC xenografts, SPARKY-X retained its unique lepidic BAC growth pattern even though it was grown in murine s.c. tissues. This property may be related to the ability of SPARKY-X to up-regulate its surfactant genes (SP-A, SP-B, and SP-D). These studies of feline BAC may allow insights into the human disease that are not possible by studying human BAC directly.

  16. Antiproliferative effect of ME3738, a derivative of soyasapogenol, on hepatocellular carcinoma cell lines in vitro and in vivo

    PubMed Central

    Ogasawara, Sachiko; Akiba, Jun; Nakayama, Masamichi; Kusano, Hironori; Yano, Hirohisa

    2016-01-01

    Soyasapogenol, an aglycon of soyasaponin, ameliorates liver injury induced by concanavalin A in mice. A derivative of soyasapogenol, 22β-methoxyolean-12-ene-3β, 24(4β)-diol (ME3738), was reported to induce the gene expression of interferon (IFN)-β in hepatitis C virus replicon cells. The effect of ME3738 on hepatocellular carcinoma (HCC) cell lines was investigated in the present study. A total of 11 HCC cell lines were cultured in medium containing 0–10 µM ME3738, and after 24, 48, or 72 h of culture, morphological observation and MTT cell growth assays were performed. Furthermore, the effects of ME3738 with or without PEG-IFN-α-2b on cell lines were investigated. Induction of apoptosis was examined on cells treated with 1 µM of ME3738 using an Annexin V assay. The effect of ME3738 (0.63 and 2.5 µM) on cell cycle progression was analyzed on two cell lines. The mice with subcutaneous tumors were divided into four groups: i) Control; ii) ME3738 alone; iii) PEG-IFN-α-2b alone and iv) ME3738+PEG-IFN-α-2b (combination). ME3738 was mixed with food (1.5 mg/g) and was taken orally for 15 days. PEG-IFN-α-2b (1,920 IU/mouse) was subcutaneously injected twice a week for two consecutive weeks. On day 15, the mice were sacrificed and the tumors were resected. A dose-dependent anti-proliferative effect was observed to various degrees in all the HCC cell lines in vitro. This inhibitory effect reached its maximal level 24 h after the treatment and the 50% inhibitory dose was between 0.8 and 2.4 µM. The combination treatment did not show a synergistic effect. Induction of apoptosis was not observed. Cell cycle arrest at S-phase was observed in two of the examined cell lines. On day 15, the tumor volume of mice receiving ME3738, PEG-IFN-α-2b, and ME3738+PEG-IFN-α-2b was 69, 30, and 33%, respectively, of the control tumor volume. ME3738 induced antiproliferative effects on the HCC cells in vitro and in vivo. The data suggested potential clinical application of ME3738

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

    PubMed

    Del Puerto, Helen L; Martins, Almir S; Milsted, Amy; Souza-Fagundes, Elaine M; Braz, Gissandra F; Hissa, Barbara; Andrade, Luciana O; Alves, Fabiana; Rajão, Daniela S; Leite, Rômulo C; Vasconcelos, Anilton C

    2011-06-30

    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.

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

    PubMed Central

    2012-01-01

    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 four well known human

  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.

    PubMed

    Feng, Sanjiang; Zhuang, Minghua; Wu, Rui

    2012-12-25

    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. Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation.

    PubMed

    Spencer, A; Granter, N

    1999-09-01

    Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both

  1. High Cytotoxicity and Apoptotic Effects of Natural Bioactive Benzofuran Derivative on the MCF-7 Breast Cancer Cell Line.

    PubMed

    Soleimani, Afsane; Asadi, Jahanbakhsh; Rostami-Charati, Faramarz; Gharaei, Roghaye

    2015-01-01

    This study was focused on evaluation of the cytotoxicity and apoptotic affects of benzofuran derivative on MCF-7 breast cancer cell line. This effective compound was isolated from the root of Petasites hybridus plant. For experiments, the MCF-7 cells were treated with several concentrations (0-500μM) of 1-(6-hydroxy-2- isopropenyl-1-benzofuran-5-yl)-1-ethanone 1 at different times. In this study, test of neutral red was also employed for cytotoxicity assay and quantity of P53, P21. Bax genes expression was analyzed using Real-Time PCR and ELISA techniques. Results show that compound 1 has cytotoxicity and apoptotic effects on Human breast cancer (Michigan Cancer Foundation-7) MCF-7 cells.

  2. Treatment Paradigms for Retinal and Macular Diseases Using 3-D Retina Cultures Derived From Human Reporter Pluripotent Stem Cell Lines

    PubMed Central

    Kaewkhaw, Rossukon; Swaroop, Manju; Homma, Kohei; Nakamura, Jutaro; Brooks, Matthew; Kaya, Koray Dogan; Chaitankar, Vijender; Michael, Sam; Tawa, Gregory; Zou, Jizhong; Rao, Mahendra; Zheng, Wei; Cogliati, Tiziana; Swaroop, Anand

    2016-01-01

    We discuss the use of pluripotent stem cell lines carrying fluorescent reporters driven by retinal promoters to derive three-dimensional (3-D) retina in culture and how this system can be exploited for elucidating human retinal biology, creating disease models in a dish, and designing targeted drug screens for retinal and macular degeneration. Furthermore, we realize that stem cell investigations are labor-intensive and require extensive resources. To expedite scientific discovery by sharing of resources and to avoid duplication of efforts, we propose the formation of a Retinal Stem Cell Consortium. In the field of vision, such collaborative approaches have been enormously successful in elucidating genetic susceptibility associated with age-related macular degeneration. PMID:27116668

  3. Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.

    PubMed Central

    Vega, Q C; Worby, C A; Lechner, M S; Dixon, J E; Dressler, G R

    1996-01-01

    The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855235

  4. Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model.

    PubMed

    Ohnishi, Hiroe; Kawasaki, Takashi; Deguchi, Tomonori; Yuba, Shunsuke

    2015-08-01

    Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.

  5. Genotype × Environment Interactions of Yield Traits in Backcross Introgression Lines Derived from Oryza sativa cv. Swarna/Oryza nivara

    PubMed Central

    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. PMID:27807437

  6. QTL Mapping of Agronomic Waterlogging Tolerance Using Recombinant Inbred Lines Derived from Tropical Maize (Zea mays L) Germplasm

    PubMed Central

    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

  7. Chordoma-derived cell line U-CH1-N recapitulates the biological properties of notochordal nucleus pulposus cells.

    PubMed

    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.

  8. Quantitative trait loci mapping for leaf length and leaf width in rice cv. IR64 derived lines.

    PubMed

    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.

  9. Effect of dehydroepiandrosterone derivatives on the activity of 5α-reductase isoenzymes and on cancer cell line PC-3.

    PubMed

    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.

  10. Frontal dermoid cyst coexisting with suprasellar craniopharyngioma: a spectrum of ectodermally derived epithelial-lined cystic lesions?

    PubMed

    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.

  11. Characterization of health-related compounds in eggplant (Solanum melongena L.) lines derived from introgression of allied species.

    PubMed

    Mennella, Giuseppe; Rotino, Giuseppe L; Fibiani, Marta; D'Alessandro, Antonietta; Francese, Gianluca; Toppino, Laura; Cavallanti, Federica; Acciarri, Nazzareno; Lo Scalzo, Roberto

    2010-07-14

    The purpose of the present study was to investigate the levels of either the nutraceutical and health-promoting compounds or the antioxidant properties of innovative eggplant (Solanum melongena L.) genotypes tolerant and/or resistant to fungi, derived from conventional and non-conventional breeding methodologies (i.e., sexual interspecific hybridization, interspecific protoplast electrofusion, androgenesis, and backcross cycles) in comparison with their allied and cultivated parents. Chemical measures of soluble refractometric residue (SRR), glycoalkaloids (solamargine and solasonine), chlorogenic acid (CA), delphinidin 3-rutinoside (D3R), total phenols (TP), polyphenoloxidase (PPO) activity, antiradical activity on superoxide anion and hydroxyl radical were carried out in raw fruit and peel of 57 eggplant advanced introgression lines (ILs), of three eggplant recurrent genotypes and of three allied species during 2005 and 2006. The majority of the ILs, obtained after several backcross cycles, showed positive characteristics with respect to the allied parents such as good levels of SRR, CA, D3R, TP, PPO activity, the scavenging activity against superoxide anion and hydroxyl radical and, in particular, significantly (p derived from the allied parents (i.e., resistance/tolerance to plant pathogen fungi) together with nutraceutical and antioxidant properties typical of the cultivated species.

  12. New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

    PubMed Central

    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

  13. A novel curcumin derivative increases the cytotoxicity of raloxifene in estrogen receptor-negative breast cancer cell lines.

    PubMed

    Taurin, Sebastien; Nimick, Mhairi; Larsen, Lesley; Rosengren, Rhonda J

    2016-01-01

    There is a need for new, safe and efficacious drug therapies for the treatment of estrogen receptor (ER)-negative breast cancers. Raloxifene and the 2nd generation curcumin derivative 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) have been shown to inhibit the growth of ER-negative breast cancer cells in vitro and in vivo. We investigated whether RL91 could enhance the growth-suppressive effects mediated by raloxifene in MDA-MB-231, MDA-MB-468, Hs578t and SkBr3 human breast cancer cell lines. The cytotoxicity was consistent across the cell lines but RL91 was more potent. EC50 values for RL91 were 1.2-2 µM while EC50 values for raloxifene were 9.6-11.2 µM. When the cells were treated with raloxifene (15 µM), RL91 (1 µM) or a combination of the two for 6-72 h, the combination treatment consistently elicited significantly greater cytotoxicity compared to all other treatments. In SkBr3 cells the combination treatment caused significantly more cells to undergo G1 arrest compared to raloxifene. In all cell lines apoptosis was synergistically induced by the combination treatment, as shown by both flow cytometery and cleaved caspase-3. Furthermore, the stress kinase p38 was increased and EFGR isoforms were decreased by both raloxifene and raloxifene + RL91. The anti-angiogenic anti-metastatic potential of raloxifene was not increased by RL91, as MDA-MB-231 cell migration and invasion as well as endothelial tube formation by HUVEC cells was not different between raloxifene (10 µM) and the combination of raloxifene + RL91. Thus, our findings provide evidence that RL91 increases the ability of raloxifene to suppress ER-negative cancer cell growth by increasing the number of apoptotic cells. The broad effect of this drug combination across a range of ER-negative breast cancer cell lines indicates that this drug combination should be explored further in order to find a safe and efficacious therapy for ER-negative breast cancer.

  14. Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture

    PubMed Central

    2012-01-01

    Introduction The development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available. Methods We derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs. Results NclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank. Conclusions The human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine. PMID:22472092

  15. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line

    PubMed Central

    TOYOHARA, YUKIYO; HASHITANI, SUSUMU; KISHIMOTO, HIROMITSU; NOGUCHI, KAZUMA; YAMAMOTO, NOBUTO; URADE, MASAHIRO

    2011-01-01

    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. PMID:22848250

  16. Inhibitory effect of vitamin D-binding protein-derived macrophage activating factor on DMBA-induced hamster cheek pouch carcinogenesis and its derived carcinoma cell line.

    PubMed

    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.

  17. Stemness in Human Thyroid Cancers and Derived Cell Lines: The Role of Asymmetrically Dividing Cancer Stem Cells Resistant to Chemotherapy

    PubMed Central

    Minsky, Noga; Morshed, Syed A.; Davies, Terry F.

    2014-01-01

    Context: Cancer stem cells (CSCs) have the ability to self-renew through symmetric and asymmetric cell division. CSCs may arise from mutations within an embryonic stem cell/progenitor cell population or via epithelial-mesenchymal transition (EMT), and recent advances in the study of thyroid stem cells have led to a growing recognition of the likely central importance of CSCs in thyroid tumorigenesis. Objective: The objectives of this study were to establish the presence of a stem cell population in human thyroid tumors and to identify, isolate, and characterize CSCs in thyroid cancer cell lines. Results: 1) Human thyroid cancers (n = 10) and thyroid cancer cell lines (n = 6) contained a stem cell population as evidenced by pluripotent stem cell gene expression. 2) Pulse-chase experiments with thyroid cancer cells identified a label-retaining cell population, a primary characteristic of CSCs, which at mitosis divided their DNA both symmetrically and asymmetrically and included a population of cells expressing the progenitor marker, stage-specific embryonic antigen 1 (SSEA-1). 3) Cells positive for SSEA-1 expressed additional stem cell markers including Oct4, Sox2, and Nanog were confirmed as CSCs by their tumor-initiating properties in vivo, their resistance to chemotherapy, and their multipotent capability. 4) SSEA-1-positive cells showed enhanced vimentin expression and decreased E-cadherin expression, indicating their likely derivation via EMT. Conclusions: Cellular diversity in thyroid cancer occurs through both symmetric and asymmetric cell division, and SSEA-1-positive cells are one form of CSCs that appear to have arisen via EMT and may be the source of malignant thyroid tumor formation. This would suggest that thyroid cancer CSCs were the result of thyroid cancer transformation rather than the source. PMID:24823711

  18. Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

    SciTech Connect

    Jinno-Oue, Atsushi; Shimizu, Nobuaki; Hamada, Nobuyuki; Wada, Seiichi; Tanaka, Atsushi; Shinagawa, Masahiko; Ohtsuki, Takahiro; Mori, Takahisa; Saha, Manujendra N.; Hoque, Ariful S.; Islam, Salequl; Kogure, Kimitaka; Funayama, Tomoo; Kobayashi, Yasuhiko

    2010-01-15

    Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated beta-galactosidase (SA-beta-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21{sup WAF1/CIP1} in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells (<1% of initially irradiated cells) could not form a colony: however, they showed a morphological phenotype consistent with cellular senescence, that is, enlarged and flattened appearance. The senescent nature of these attached cells was further indicated by staining for SA-beta-gal. The mean telomere length was not changed after irradiation with C-ions. Phosphorylation of p53 at serine 15 as well as the expression of p21{sup WAF1/CIP1} was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

  19. The effects of ultraviolet light on aspects of DNA metabolism in cell lines derived from plodia interpunctella and Trichoplusia ni

    SciTech Connect

    Styer, S.C.

    1991-01-01

    Insect cells are significantly more resistant to the lethal effects of 254 nm ultraviolet light (UV) than mammalian cells. The predominant photoproduct produced by UV is the (5-6) cyclobutyl pyrimidine dimer. There is controversy whether this lesion, or another, the pyrimidine (6-4) pyrimidone, is responsible for the biological effects of UV. Insect cells contain a photolyase which selectively removes the (5-6), but not the (6-4) lesion, so that the relative roles of these lesions can be studied. Insect cell lines derived from the cabbage looper and the Indian meal moth were exposed to UV and analyzed for their ability to incorporate [sup 3]H-thymidine. After exposure, cells from the Indian meal moth exhibited a rapid and prolonged depression in the rate of thymidine incorporation, whereas cells from the cabbage looper showed only a slight drop in incorporation and a rapid recovery. The extent of depression in thymidine incorporation was not correlated to the amount of cell killing by UV in these cell lines. Blockage of fork progression was correlated to the depression in thymidine incorporation. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation or cell killing, indicating that although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions, such as the (6-4) photoproduct, may play a role. In addition, activation of alternative sites of replicon initiation appeared to correlate with the depression in thymidine incorporation and the excision capabilities in these cells. The resistance to UV in these insect cells compared to mammalian cells may be due to their ability to rapidly remove the (5-6) lesion, which is the critical lesion in these insect cells.

  20. Expression of brain-derived neurotrophic factor and TrkB in the lateral line system of zebrafish during development.

    PubMed

    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.

  1. Associations of corticosterone and testosterone with alcohol drinking in F2 populations derived from AA and ANA rat lines.

    PubMed

    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.

  2. Unique Polycomb Gene Expression Pattern in Hodgkin’s Lymphoma and Hodgkin’s Lymphoma-Derived Cell Lines

    PubMed Central

    Dukers, Danny F.; van Galen, Joost C.; Giroth, Cindy; Jansen, Patty; Sewalt, Richard G.A.B.; Otte, Arie P.; Kluin-Nelemans, Hanneke C.; Meijer, Chris J.L.M.; Raaphorst, Frank M.

    2004-01-01

    Human Polycomb-group (PcG) genes play a crucial role in the regulation of embryonic development and regulation of the cell cycle and hematopoiesis. PcG genes encode proteins that form two distinct PcG complexes, involved in maintenance of cell identity and gene silencing patterns. We recently showed that expression of the BMI-1 and EZH2 PcG genes is separated during normal B-cell development in germinal centers, whereas Hodgkin/Reed-Sternberg (H/RS) cells co-express BMI-1 and EZH2. In the current study, we used immunohistochemistry and immunofluorescence to determine whether the binding partners of these PcG proteins are also present in H/RS cells and H/RS-derived cell lines. PcG expression profiles were analyzed in combination with expression of the cell cycle inhibitor p16INK4a, because experimental model systems indicate that p16 is a downstream target of Bmi-1. We found that H/RS cells and HL-derived cell lines co-express all core proteins of the two known PcG complexes, including BMI-1, MEL-18, RING1, HPH1, HPC1, and -2, EED, EZH2, YY1, and the HPC2 binding partner, CtBP. Expression of HPC1 has not been found in normal mature B cells and other malignant lymphomas of B-cell origin, suggesting that the PcG expression profile of H/RS is unique. In contrast to Bmi-1 transgenic mice where p16INK4a is down-regulated, 27 of 52 BMI-1POS cases of HL revealed strong nuclear expression of p16INK4a. We propose that abnormal expression of BMI-1 and its binding partners in H/RS cells contributes to development of HL. However, abnormal expression of BMI-1 in HL is not necessarily associated with down-regulation of p16INK4a. PMID:14982841

  3. Mass spectrometry data processing using zero-crossing lines in multi-scale of Gaussian derivative wavelet

    PubMed Central

    Nguyen, Nha; Huang, Heng; Oraintara, Soontorn; Vo, An

    2010-01-01

    Motivation: Peaks are the key information in mass spectrometry (MS) which has been increasingly used to discover diseases-related proteomic patterns. Peak detection is an essential step for MS-based proteomic data analysis. Recently, several peak detection algorithms have been proposed. However, in these algorithms, there are three major deficiencies: (i) because the noise is often removed, the true signal could also be removed; (ii) baseline removal step may get rid of true peaks and create new false peaks; (iii) in peak quantification step, a threshold of signal-to-noise ratio (SNR) is usually used to remove false peaks; however, noise estimations in SNR calculation are often inaccurate in either time or wavelet domain. In this article, we propose new algorithms to solve these problems. First, we use bivariate shrinkage estimator in stationary wavelet domain to avoid removing true peaks in denoising step. Second, without baseline removal, zero-crossing lines in multi-scale of derivative Gaussian wavelets are investigated with mixture of Gaussian to estimate discriminative parameters of peaks. Third, in quantification step, the frequency, SD, height and rank of peaks are used to detect both high and small energy peaks with robustness to noise. Results: We propose a novel Gaussian Derivative Wavelet (GDWavelet) method to more accurately detect true peaks with a lower false discovery rate than existing methods. The proposed GDWavelet method has been performed on the real Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight (SELDI-TOF) spectrum with known polypeptide positions and on two synthetic data with Gaussian and real noise. All experimental results demonstrate that our method outperforms other commonly used methods. The standard receiver operating characteristic (ROC) curves are used to evaluate the experimental results. Availability: http://ranger.uta.edu/∼heng/MS/GDWavelet.html or http://www.naaan.org/nhanguyen/archive.htm Contact: heng

  4. Anticancer effects of cinnamic acid in lung adenocarcinoma cell line h1299-derived stem-like cells.

    PubMed

    Huang, Yanyan; Zeng, Fang; Xu, Liyun; Zhou, Jihang; Liu, Xiaoguang; Le, Hanbo

    2013-01-01

    Lung cancer is a lethal solid tumor with poor prognosis because of its high metastasis and resistance to current therapies. Recently, cancer stem cells (CSCs) were suggested to be major contributors to tumorigenicity and cancer relapse. However, therapeutic targets for lung cancer-related CSCs remain undetermined. The objective of the current study was to investigate whether cinnamic acid (CINN) exerts an antitumor activity against sphere-derived lung CSCs. In this study, CSCs were isolated from the non-small cell lung cancer cell line H1299 as tumor spheres under CSC-selective conditions, and found to have increased tumorigenicity, chemoresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared with parental cells. These observations are consistent with the notion that CSCs are tumorigenic, display the ability to self-renew, and generate differentiated progeny that constitute the majority of cells in tumors. Treatment of sphere-derived stem cells with CINN could diminish their CSC-like abilities by decreasing their proliferation and invasive abilities and facilitating their differentiation into CD133-negative cells. Furthermore, CINN treatment increased the sensitivity of CSCs to chemotherapeutic drugs through apoptosis. Of note, xenotransplantation experiments revealed that CINN combined with cisplatin had a synergistic effect in inhibiting the tumorigenicity of CSCs. In summary, our study clearly revealed the presence of a population of sphere-forming cells with stem-like properties among H1299 cells and CINN can attenuate CSC properties of this stem-like cell population. The potential of CINN should be verified further in future studies of anti-CSC therapy.

  5. Differentiation of Meloidogyne incognita and M. arenaria novel resistance phenotypes in Lycopersicon peruvianum and derived bridge-lines.

    PubMed

    Veremis, J C; Roberts, P A

    1996-10-01

    Lycopersicon peruvianum PI 270435 clone 2R2 and PI 126443 clone 1MH were crossed reciprocally with three L. esculentum-L. peruvianum bridge-lines. The incongruity barrier between the two plant species was overcome; F1 progeny were obtained from crosses between four parental combinations without embryo-rescue culture. Hybridity was confirmed by leaf and flower morphology and by the production of nematode-resistant F1 progeny on homozygous susceptible parents. Clones of the five F1 bridgeline hybrids were highly resistant to Mi-avirulent root-knot nematode (Meloidogyne incognita) at both 25°C and 30°C soil temperatures. However, only clones from PI 270435-3MH and PI 126443-1MH, and hybrids from PI 126443-1MH, were resistant to Mi-virulent M. incognita isolates at high soil temperature. Clones and hybrids from PI 270435-2R2 were not resistant to two Mi-virulent M. incognita isolates at high soil temperature. A source of heat-stable resistance was identified in bridge-line EPP-2, and was found to be derived from L. peruvianum LA 1708. Accessions of the L. peruvianum 'Maranon races', LA 1708 and LA 2172, and bridge-line EPP-2, segregated for heat-stable resistance to Mi-avirulent M. incognita, but were susceptible to Mi-virulent M. incognita isolates. Clone LA 1708-I conferred heat-stable resistance to M. arenaria isolate W, which is virulent to heat-stable resistance genes in L. peruvianum PI 270435-2R2, PI 270435-3MH, and PI 126443-1MH. Clone LA 1708-I has a distinct heat-stable factor for resistance to Mi-avirulent M. arenaria isolate W, for which the gene symbol Mi-4 is proposed. A Mi-virulent M. arenaria isolate Le Grau du Roi was virulent on all Lycopersicon spp. accessions tested, including those with novel resistance genes.

  6. Effect of cell-derived growth factors and cytokines on the clonal outgrowth of EBV-infected B cells and established lymphoblastoid cell lines.

    PubMed

    Ifversen, P; Zhang, X M; Ohlin, M; Zeuthen, J; Borrebaeck, C A

    1993-07-01

    Epstein-Barr virus (EBV) is a potent inducer of polyclonal B lymphocyte proliferation and is widely used as a tool for the establishment of B cell lines producing human monoclonal antibodies. However, because of low transformability, low clonability, and the inherent instability of EBV-infected B cells, valuable antibody-producing B cells are often lost during this procedure. We have here examined various cell-derived cytokines for their ability to enhance both the cellular outgrowth of newly infected B cells and the clonability of infected B cells and lymphoblastoid cell lines. Our results show that the murine thymoma cell line EL-4 is superior to peripheral blood mononuclear cells in both cellular outgrowth and cloning experiments, whereas monocyte-derived factors and monocyte cell lines were less capable than peripheral blood mononuclear cells in enhancing cellular outgrowth and cloning. Furthermore, the human T cell hybridoma cell line MP6 that secretes a B cell growth and differentiation factor, recently identified as an isoform of thioredoxin, is also capable of stimulating EBV-infected B cells and lymphoblastoid cell lines. Co-cultivation of EBV-infected B cells with MP6 cells significantly enhanced the cloning efficiency at the 1 cell/well level. The present results also suggest that one potential role of the MP6-derived thioredoxin could be the up regulation of IL-6 receptor expression in EBV-infected B cells.

  7. An occult hepatitis B-derived hepatoma cell line carrying persistent nuclear viral DNA and permissive for exogenous hepatitis B virus infection.

    PubMed

    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.

  8. Exposure to 1800 MHz radiofrequency electromagnetic radiation induces oxidative DNA base damage in a mouse spermatocyte-derived cell line.

    PubMed

    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.

  9. Novel 4-anilinoquinazoline derivatives featuring an 1-adamantyl moiety as potent EGFR inhibitors with enhanced activity against NSCLC cell lines.

    PubMed

    Yu, Haiqing; Li, Yanxia; Ge, Yang; Song, Zhendong; Wang, Changyuan; Huang, Shanshan; Jin, Yue; Han, Xu; Zhen, Yuhong; Liu, Kexin; Zhou, Youwen; Ma, Xiaodong

    2016-03-03

    With the aim of overcoming gefitinib resistance, a series of novel quinazoline derivatives bearing an adamantyl group on the aniline ring were synthesized as potent epidermal growth factor receptor (EGFR) inhibitors. Most of these analogues are comparable to gefitinib in their ability to inhibit non-small cell lung cancer (NSCLC) cell lines, and several also exhibited significantly enhanced anti-tumor potency. Specifically, compound 3d, with an IC50 value of 2.06 μM against A431 cells with the wild-type EGFR and of 0.009 μM against the gefitinib-sensitive cells, displayed approximately 5-fold higher potency than the lead compound to inhibit the cells harboring the EGFR(T790M) mutant. In addition, the molecular simulation and Western blot analysis results also indicated that these compounds effectively interfered with the EGFR(T790M) activity, and may serve as a new alternative structure to develop more effective antitumor agents.

  10. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

    PubMed

    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.

  11. Synthesis, analysis and cytotoxic evaluation of some hydroxypyridinone derivatives on HeLa and K562 cell lines

    PubMed Central

    Saghaie, L.; Sadeghi-Aliabadi, H.; Ashaehshoar, M.

    2013-01-01

    A range of iron bidentae ligands containing the chelating moiety 3- hydroxypyridin-4-ones (HPOs) have been synthesized via a single or a three-step synthetic pathway. In the single-step reaction, maltol was directly reacted by suitable primary amine and in the second synthetic method; benzylated maltol was reacted with related amines to give 1-substuted-2-methyl-3-benzyloxypyridin-4-one derivatives. Finally, removal of the benzyl group under acidic conditions was performed by catalytic hydrogenation to yield the favored bidentate chelators as HCl salt. The partition coefficient of the free ligands and their iron (III) complexes between an aqueous phase buffered at pH 7.4 and 1-octanol were also determined. The cytotoxic effects of these iron chelators against HeLa and K562 cell lines were evaluated using MTT assay and the results showed that cytotoxicity was closely related to the lipophilicity of compounds so that the most lipophilic compound (4g) revealed the highest activity and compound 4e as a more hydrophilic agent (Kpart; 0.05) showed the lowest cytotoxic effect. PMID:24019828

  12. Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma.

    PubMed

    Xiong, Ye; Liu, Liyun; Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

    2017-02-12

    As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion.

  13. Novel Quinazoline Derivatives Bearing Various 4-Aniline Moieties as Potent EGFR Inhibitors with Enhanced Activity Against NSCLC Cell Lines.

    PubMed

    Wang, Changyan; Sun, Yajun; Zhu, Xingqi; Wu, Bin; Wang, Qiao; Zhen, Yuhong; Shu, Xiaohong; Liu, Kexin; Zhou, Youwen; Ma, Xiaodong

    2016-04-01

    A class of novel quinazoline derivatives bearing various C-4 aniline moieties was synthesized and biologically evaluated as potent epidermal growth factor receptor (EGFR) inhibitors for intervention of non-small-cell lung cancer (NSCLC). Most of these inhibitors are comparable to gefitinib in inhibiting these cancer cell lines, and several of them even displayed superior inhibitory activity. In particular, analogue 5b with an IC50 of 0.10 μm against the EGFR wild-type A431 cells and 5c with an IC50 of 0.001 μm against the gefitinib-sensitive HCC827 cells (EGFR del E746-A750) was identified as highly active EGFR inhibitors. It was also significant that the discovered analogue 2f, not only has high potency against the gefitinib-sensitive cells (IC50 = 0.031 μm), but also possesses remarkably improved activity against the gefitinib-resistant cells. In addition, the enzymatic assays and the Western blot analysis for evaluating the effects of the typical inhibitors indicated that these molecules strongly interfere with the EGFR target.

  14. Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor*

    PubMed Central

    Liu, Zhong-wei; Li, Wen-qiang; Wang, Jun-kui; Ma, Xian-cang; Liang, Chen; Liu, Peng; Chu, Zheng; Dang, Yong-hui

    2014-01-01

    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. PMID:25471830

  15. Zirconium oxide ceramic foam: a promising supporting biomaterial for massive production of glial cell line-derived neurotrophic factor.

    PubMed

    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.

  16. The effect of line damping, magneto-optics and parasitic light on the derivation of sunspot vector magnetic fields

    NASA Technical Reports Server (NTRS)

    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.

  17. Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells.

    PubMed

    Vianney, John-Mary; Miller, Damon A; Spitsbergen, John M

    2014-11-07

    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.

  18. Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines.

    PubMed

    Rungsiwiwut, Ruttachuk; Ingrungruanglert, Praewphan; Numchaisrika, Pranee; Virutamasen, Pramuan; Phermthai, Tatsanee; Pruksananonda, Kamthorn

    2016-01-01

    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.

  19. Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins.

    PubMed

    Hashimoto, Yoshi; Zhang, Sheng; Zhang, Shiying; Chen, Yun-Ru; Blissard, Gary W

    2012-04-24

    After publication we discovered an error in the identification of the origin of the cell line reported in our article in BMC Biotechnology (2010, 10:50), entitled "Ao38, a new cell line from eggs of the black witch moth, Ascalapha odorata (Lepidoptera: Noctuidae), is permissive for AcMNPV infection and produces high levels of recombinant proteins". Upon analysis of primary A. odorata cultures, we found that they were contaminated with cells of Trichoplusia ni origin. The origin of the Ao38 cell line was determined as T. ni using three marker genes and the Ao38 cell line was renamed BTI-Tnao38. References to the origin of the cell line as Ascalapha odorata should be replaced with "a cell line of Trichoplusia ni origin". The absence of TNCL virus detection in the BTI-Tnao38 (Ao38) cell line was confirmed using a highly sensitive RT-PCR protocol capable of detecting TNCL virus RNA at approximately 0.018 copies/cell. Because of these observations, we have revised the title of the original article to "Correction: BTI-Tnao38, a new cell line derived from Trichoplusia ni, is permissive for AcMNPV infection and produces high levels of recombinant proteins" and two additional authors were added to reflect their contributions to the analysis of this cell line.

  20. Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Cell Line Derived 5-FU and Irinotecan Drug-Sensitivity Profiles Evaluated in Adjuvant Colon Cancer Trial Data

    PubMed Central

    Delorenzi, Mauro; Jensen, Thomas; Jensen, Peter Buhl; Bosman, Fred; Tejpar, Sabine; Roth, Arnaud; Brunner, Nils; Hansen, Anker; Knudsen, Steen

    2016-01-01

    Purpose This study evaluates whether gene signatures for chemosensitivity for irinotecan and 5-fluorouracil (5-FU) derived from in vitro grown cancer cell lines can predict clinical sensitivity to these drugs. Methods To test if an irinotecan signature and a SN-38 signature could identify 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 patients enrolled in the PETACC-3 prospective randomized clinical trial. A 5-FU profile developed similarly was assessed by comparing the PETACC-3 cohort with a cohort of 359 stage II colon cancer patients who underwent surgery but received no adjuvant therapy. Results There was no statistically significant association between the irinotecan or SN-38 profiles and benefit from irinotecan. The 5-FU sensitivity profile showed a statistically significant association with relapse free survival (RFS) (hazard ratio (HR) = 0.54 (0.41–0.71), p<1e-05) and overall survival (HR = 0.47 (0.34–0.63), p<1e-06) in the PETACC-3 subpopulation. The effect of the 5-FU profile remained significant in a multivariable Cox Proportional Hazards model, adjusting for several relevant clinicopathological parameters. No statistically significant effect of the 5-FU profile was observed in the untreated cohort of 359 patients (relapse free survival, p = 0.671). Conclusion The irinotecan predictor had no predictive value. The 5-FU predictor was prognostic in stage III patients in PETACC-3 but not in stage II patients with no adjuvant therapy. This suggests a potential predictive ability of the 5-FU sensitivity profile to identify colon cancer patients who may benefit from 5-FU, however, any biomarker predicting benefit for adjuvant 5-FU must be rigorously evaluated in independent cohorts. Given differences

  2. Characterization of Quantitative Trait Loci Controlling Genetic Variation for Preharvest Sprouting in Synthetic Backcross-Derived Wheat Lines

    PubMed Central

    Imtiaz, Muhammad; Ogbonnaya, Francis C.; Oman, Jason; van Ginkel, Maarten

    2008-01-01

    Aegilops tauschii, the wild relative of wheat, has stronger seed dormancy, a major component of preharvest sprouting resistance (PHSR), than bread wheat. A diploid Ae. tauschii accession (AUS18836) and a tetraploid (Triticum turgidum L. ssp. durum var. Altar84) wheat were used to construct a synthetic wheat (Syn37). The genetic architecture of PHS was investigated in 271 BC1F7 synthetic backcross lines (SBLs) derived from Syn37/2*Janz (resistant/susceptible). The SBLs were evaluated in three environments over 2 years and PHS was assessed by way of three measures: the germination index (GI), which measures grain dormancy, the whole spike assay (SI), which takes into account all spike morphology, and counted visually sprouted seeds out of 200 (VI). Grain color was measured using both Chroma Meter- and NaOH-based approaches. QTL for PHSR and grain color were mapped and their additive and epistatic effects as well as their interactions with environment were estimated by a mixed linear-model approach. Single-locus analysis following composite interval mapping revealed four QTL for GI, two QTL for SI, and four QTL for VI on chromosomes 3DL and 4AL. The locus QPhs.dpiv-3D.1 on chromosome 3DL was tightly linked to the red grain color (RGC) at a distance of 5 cM. The other locus on chromosome 3D, “QPhs.dpiv-3D.2” was independent of RGC locus. Two-locus analysis detected nine QTL with main effects and 18 additive × additive interactions for GI, SI, and VI. Two of the nine main effects QTL and two epistatic QTL showed significant interactions with environments. Both additive and epistatic effects contributed to phenotypic variance in PHSR and the identified markers are potential candidates for marker-assisted selection of favorable alleles at multiple loci. SBLs derived from Ae. tauschii proved to be a promising tool to dissect, introgress, and pyramid different PHSR genes into adapted wheat genetic backgrounds. The enhanced expression of PHS resistance in SBLs enabled

  3. Detection of cell surface and intracellular antigens by human monoclonal antibodies. Hybrid cell lines derived from lymphocytes of patients with malignant melanoma

    PubMed Central

    1983-01-01

    This study represents an initial attempt to analyze the humoral immune reactions of patients with malignant melanoma by hybridoma methodology. Using lymphocytes from regional lymph nodes, peripheral blood and tumor infiltrates, 158 fusions were performed with SKO-007 (human myeloma line), LICR-LON-HMy2 (LICR-2), GM 4672 (human lymphoblastoid lines), or NS-1 (mouse myeloma line). Fusion of lymph node lymphocytes with NS-1 resulted in a 3-4 times higher frequency of clones than fusion with LICR-2, and a 10 times higher frequency than fusion with SKO-007 or GM 4672. In the case of peripheral blood lymphocytes, fusion with NS-1 gave greater than 25 times higher frequency of clones than fusion with LICR-2 or SKO-007. Production of human mu, gamma, or alpha heavy chains was detected in 50-80% of wells containing growing clones, and the levels of immunoglobulin ranged from 0.3 micrograms to 40 micrograms/ml. NS-1-derived clones could be easily subcultured, while LICR-2 and SKO-007 clones grew more slowly on subculturing. In this study, Ig secretion appeared to be a more stable property of LICR-2- derived clones than NS-1-derived clones. A panel of 20 human cancer cell lines was used to screen 771 Ig-secreting cultures for antibody to cell surface or intracellular antigens. Reactivity with cell surface antigens was found infrequently (6 cultures), whereas reactivity with intracellular antigens was more common (27 cultures). A new cell surface antigen with properties of a glycolipid was defined with an IgM monoclonal antibody secreted by a tetraploid cell derived from a fusion of LICR-2 with lymphocytes from the axillary lymph node of a patient with melanoma. The hybrid cell line has been subcloned four times and secretes 5 micrograms IgM/ml. The antigen detected by this IgM antibody was found on 5 of 23 melanoma cell lines and 12 of 30 epithelial cancer cell lines. No reactions were found with 11 cultures derived from normal cells. Stable cell lines secreting human

  4. Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSC line H266.

    PubMed

    Marthaler, Adele G; Tubsuwan, Alisa; Schmid, Benjamin; Poulsen, Ulla B; Engelbrecht, Alexander F; Mau-Holzmann, Ulrike A; Hyttel, Poul; Nielsen, Troels T; Nielsen, Jørgen E; Holst, Bjørn

    2016-01-01

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease primarily affecting the cerebellum. Very little is known about the molecular mechanisms underlying the disease and, to date, no cure or treatment is available. We have successfully generated bona fide induced pluripotent stem cell (iPSC) lines of SCA2 patients in order to study a disease-specific phenotype. Here, we demonstrate the gene correction of the iPSC line H266 clone 10 where we have exchanged the expanded CAG repeat of the ATXN2 gene with the normal length found in healthy alleles. This gene corrected cell line will provide the ideal control to model SCA2 by iPSC technology.

  5. Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSC line H271.

    PubMed

    Marthaler, Adele G; Schmid, Benjamin; Tubsuwan, Alisa; Poulsen, Ulla B; Engelbrecht, Alexander F; Mau-Holzmann, Ulrike A; Hyttel, Poul; Nielsen, Jørgen E; Nielsen, Troels T; Holst, Bjørn

    2016-01-01

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease primarily affecting the cerebellum. Very little is known about the molecular mechanisms underlying the disease and, to date, no cure or treatment is available. We have successfully generated bona fide induced pluripotent stem cell (iPSC) lines of SCA2 patients in order to study a disease-specific phenotype. Here, we demonstrate the gene correction of the iPSC line H271 clone 1 where we have exchanged the expanded CAG repeat of the ATXN2 gene with the normal length found in healthy alleles. This gene corrected cell line will provide the ideal control to model SCA2 by iPSC technology.

  6. Generation of an isogenic, gene-corrected control cell line of the spinocerebellar ataxia type 2 patient-derived iPSC line H196.

    PubMed

    Marthaler, Adele G; Schmid, Benjamin; Tubsuwan, Alisa; Poulsen, Ulla B; Engelbrecht, Alexander F; Mau-Holzmann, Ulrike A; Hyttel, Poul; Nielsen, Jørgen E; Nielsen, Troels T; Holst, Bjørn

    2016-01-01

    Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease primarily affecting the cerebellum. Very little is known about the molecular mechanisms underlying the disease and, to date, no cure or treatment is available. We have successfully generated bona fide induced pluripotent stem cell (iPSC) lines of SCA2 patients in order to study a disease-specific phenotype. Here, we demonstrate the gene correction of the iPSC line H196 clone 7 where we have exchanged the expanded CAG repeat of the ATXN2 gene with the normal length found in healthy alleles. This gene corrected cell line will provide the ideal control to model SCA2 by iPSC technology.

  7. Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture.

    PubMed

    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.

  8. Imipramine activates glial cell line-derived neurotrophic factor via early growth response gene 1 in astrocytes.

    PubMed

    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.

  9. Comparative study on the cytotoxic effects of benzalkonium chloride on the Wong-Kilbourne derivative of Chang conjunctival and IOBA-NHC cell lines

    PubMed Central

    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

  10. 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

    PubMed Central

    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

  11. Deriving the Extinction to Young Stellar Objects using [Fe II] Near-infrared Emission Lines: Prescriptions from GIANO High-resolution Spectra

    NASA Astrophysics Data System (ADS)

    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.

  12. A Sclerostin super-producer cell line derived from the human cell line SaOS-2: a new tool for the study of the molecular mechanisms driving Sclerostin expression.

    PubMed

    Pérez-Campo, Flor M; Sañudo, Carolina; Delgado-Calle, Jesús; Arozamena, Jana; Zarrabeitia, María T; Riancho, José A

    2014-08-01

    Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.

  13. Derivation of HVR1, HVR2 and HVR3 human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis (PGD) for monogenic disorder.

    PubMed

    Hmadcha, Abdelkrim; Aguilera, Yolanda; Lozano-Arana, Maria Dolores; Mellado, Nuria; Sánchez, Javier; Moya, Cristina; Sánchez-Palazón, Luis; Palacios, Jose; Antiñolo, Guillermo; Soria, Bernat

    2016-05-01

    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.

  14. The development of frost tolerance and DHN5 protein accumulation in barley (Hordeum vulgare) doubled haploid lines derived from Atlas 68 x Igri cross during cold acclimation.

    PubMed

    Kosová, Klára; Tom Prásil, Ilja; Prásilová, Pavla; Vítámvás, Pavel; Chrpová, Jana

    2010-03-15

    The dynamics of a long-term cold acclimation (CA) was studied in spring barley cultivar Atlas 68, winter barley cultivar Igri and a set of doubled haploid (DH) lines derived from an Atlas 68xIgri cross. The aim was to evaluate the effect of plant development on the ability to induce frost tolerance (FT) and to accumulate dehydrin 5 (DHN5) during CA. The plant developmental stage was evaluated by phenological development of the shoot apex and by determination of days to heading after a certain period of CA. FT was determined by direct frost tests. Plant winter survival was also determined. DHN5 was evaluated by densitometric analysis of protein gel blots. Cold led to the induction of increased FT and to the accumulation of DHN5 in both spring and winter lines. However, with the progression of CA, differences between the growth habits occurred as the winter lines were able to maintain increased FT and DHN5 levels for a significantly longer period of time than the spring lines. After vegetative/reproductive transition, a significant decrease in DHN5 accumulation was found in all lines; however, a discrepancy between the acquired FT level and DHN5 accumulation in vernalized winter barley plants was found. A correlation between DHN5 accumulation and plant winter survival was found when the studied lines were differentiated according to their developmental stage and DHN5 level. Possible explanations for these phenomena are provided.

  15. Relationships among the slopes of lines derived from various data analysis techniques and the associated correlation coefficient

    NASA Technical Reports Server (NTRS)

    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.

  16. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

    PubMed

    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.

  17. Surface glycoproteins of an African henipavirus induce syncytium formation in a cell line derived from an African fruit bat, Hypsignathus monstrosus.

    PubMed

    Krüger, Nadine; Hoffmann, Markus; Weis, Michael; Drexler, Jan Felix; Müller, Marcel Alexander; Winter, Christine; Corman, Victor Max; Gützkow, Tim; Drosten, Christian; Maisner, Andrea; Herrler, Georg

    2013-12-01

    Serological screening and detection of genomic RNA indicates that members of the genus Henipavirus are present not only in Southeast Asia but also in African fruit bats. We demonstrate that the surface glycoproteins F and G of an African henipavirus (M74) induce syncytium formation in a kidney cell line derived from an African fruit bat, Hypsignathus monstrosus. Despite a less broad cell tropism, the M74 glycoproteins show functional similarities to glycoproteins of Nipah virus.

  18. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

    PubMed Central

    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. PMID:27070801

  19. Synthesis of novel 1,8-acridinediones derivatives: Investigation of MDR reversibility on breast cancer cell lines T47D and tamoxifen-resistant T47D

    PubMed Central

    Moallem, S.A.; Dehghani, N.; Mehri, S.; Shahsavand, Sh.; Alibolandi, M.; Hadizadeh, F.

    2015-01-01

    Multi drug resistance (MDR) is a serious obstacle in the management of breast cancer. Therefore, overcoming MDR using novel anticancer agents is a top priority for medicinal chemists. It was found that dihydropyridines lacking calcium antagonistic activity (e.g acridinediones) possess MDR modifier potency. In this study, the capability of four novel acridine-1,8-diones derivatives 3a-d were evaluated as MDR reversing agents. In addition, the relationship between structural properties and biological effects of synthesized compounds was discussed. In vitro cytotoxicity of acridine-1,8-diones 3a-d derivatives in combination with doxorubicin (DOX) on T47D and tomoxifen-resistant T47D (TAMR-6) breast cancer cell lines were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Drug resistant index (DRI), which is equal to the ratio of IC50 in drug-resistant cells over IC50 in drug-sensitive cells, was calculated for each substance. Flowcytometry experiments were also implemented to distinguish cells undergoing apoptosis from those undergoing necrosis. The results from MTT and flowcytometry experiments indicated that 1 nM 3c derivative along with DOX significantly (P<0.05) increased the DOX cytotoxicity in T47D and TAMR-6 breast cancer cell lines. Synthesized compounds 3a and 3b also at concentrations of 1 nM with DOX significantly increased the cytotoxicity of DOX on T47D and TAMR-6 breast cancer cell lines. Meanwhile, 3d derivative with DOX did not exhibit good synergistic effect on cytotoxic activity of DOX, and slightly increased DOX cytotoxicity in both cell lines. Our results proposed that 3c may be an attractive lead compound for further development as a chemotherapeutic agent for MDR breast cancer therapy in combination with routine chemotherapeutic agents such as DOX. PMID:26600848

  20. A novel fish cell line derived from the brain of Chinese perch Siniperca chuatsi: development and characterization.

    PubMed

    Fu, X; Li, N; Lai, Y; Luo, X; Wang, Y; Shi, C; Huang, Z; Wu, S; Su, J

    2015-01-01

    In this study, a continuous cell line (named as CPB) was established from Siniperca chuatsi brain and has been subcultured >140 times. CPB cell line predominantly consisted of fibroblast-like cells that could grow better in Leibovitz's L-15 supplemented with 10% foetal bovine serum at 28° C. Polymerase chain reaction amplification of 18s recombinant (r)RNA confirmed the origin of this cell line from S. chuatsi. The CPB cell line was cryopreserved at different passage levels and revived successfully with 80-90% survival. The cell line was further characterized by chromosome number and transfection. The CPB cells were highly susceptible to infectious spleen and kidney necrosis virus (ISKNV) with a titre of 6·58-6·62 log TCID50 ml(-1) and numerous ISKNV particles were observed in the cytoplasm by transmission electron microscopy. At the same time, ISKNV infection was confirmed by reverse transcriptase polymerase chain reaction, immunodot blot and individual challenge experiments. The development and characterization of a new brain cell line from S. chuatsi were described in this study and it could be used as an in vitro tool for propagation of ISKNV and gene expression studies.

  1. Stars with discrepant v sin i as derived from the Ca II 3933 and Mg II 4481 Å lines. VI. HD 199892 — an SB2 spectroscopic binary

    NASA Astrophysics Data System (ADS)

    Zverko, J.; Romanyuk, I.; Iliev, I.; Kudryavtsev, D.; Stateva, I.; Semenko, E.

    2017-01-01

    We analyzed the spectra of a well known SB1 binary HD199892 for which the projected rotational velocity v sin i, introduced in the literature, significantly differs when determined from the lines of Ca II at 3933 Å and ofMg II at 4481 Å. Contrary to the former findings, we discovered the signs of spectral lines of a companion star in the profile of Hβ as well as weak metallic lines in the high resolution high S/N spectra covering the most of the visual region. We estimated the secondary star to be a main sequence A4V star with a mass of 2.2 M ⊙ and derived its radial velocity which resulted in the mass of the primary M = 4.6 M ⊙. Short sections of the spectra in the Mg II 4481 Å and Ca II 3933 Å regions are analyzed as well.

  2. Comparison between observed and theoretical O IV line ratios in the UV/EUV solar spectrum as derived by SUMER, CDS and EIS

    NASA Astrophysics Data System (ADS)

    Giunta, A. S.; Fludra, A.; O'Mullane, M. G.; Summers, H. P.

    2012-02-01

    Aims: The joint use of SoHO Solar Ultraviolet Measurement of Emitted Radiation (SUMER), Coronal Diagnostic Spectrometer (CDS) and Hinode Extreme-ultraviolet Imaging Spectrometer (EIS) allow observation of several O iv line ratios, useful for temperature plasma diagnostics. Accurate atomic data are required to avoid interpretation errors in deriving the electron temperature from these ratios. Muglach et al. (2010) found that the measured ratio I(787.72 Å)/I(279.93 Å) is lower than the predicted value by a factor 2-5. Here the predicted value for this ratio is revised using updated atomic data. A comparison with other observed O iv line ratios is shown and the electron temperature is derived. Methods: The analysis is based on new observations made during the observational campaign of April 2009 and including three O iv multiplets. The theoretical ratios have been derived using the Atomic Data and Analysis Structure (ADAS) database and include comparison with the most recent calculations available in the literature. Results: The discrepancy for the O iv I(787.72 Å)/I(279.93 Å) ratio has been solved by adding transitions involving higher excited levels, which have been omitted in previous atomic models. This results in a decrease of the theoretical line ratio, providing electron temperatures in the range of log T = 5.17-5.39, close to the temperature expected from a plasma in ionisation equilibrium.

  3. Potential of on-line visible and near infrared spectroscopy for measurement of pH for deriving variable rate lime recommendations.

    PubMed

    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.

  4. Establishment and characterization of an astroglial cell line derived from the brain of half-smooth tongue sole (Cynoglossus semilaevis)

    PubMed Central

    WANG, Tian-Zi; SUN, Ai; WANG, Na; CUI, Zhong-Kai; CHEN, Song-Lin; SHA, Zhen-Xia

    2015-01-01

    An astroglial cell line was established from the brain of half smooth tongue sole (Cynoglossus semilaevis) and was designated as CSAC. CSAC shows the morphological homogeneity of epithelial cells. The cell identity was tested by the presence of glial fibrillary acidic protein (GFAP), which was revealed by RT-PCR and immunofluorescence. The cell line was optimally maintained at 24 °C in minimum essential medium supplemented with HEPES, antibiotics, 20% fetal bovine serum, 2-Mercaptoethanol (2-Me) and basic fibroblast growth factor. Chromosome analysis revealed that the CSAC cells maintained a normal diploid chromosome number (2n=42). The fluorescent signals were observed in CSAC after the cells were transfected with green fluorescent protein (GFP) reporter plasmids. The CSAC cell line may serve as a valuable tool for studies on the potential functions of fish astroglial cells. PMID:26452695

  5. A glial cell line-derived neurotrophic factor-secreting clone of the Schwann cell line SCTM41 enhances survival and fiber outgrowth from embryonic nigral neurons grafted to the striatum and to the lesioned substantia nigra.

    PubMed

    Wilby, M J; Sinclair, S R; Muir, E M; Zietlow, R; Adcock, K H; Horellou, P; Rogers, J H; Dunnett, S B; Fawcett, J W

    1999-03-15

    We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson's disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.

  6. Morphological and molecular effects of 20-hydroxyecdysone and its agonist tebufenozide on CF-203, a midgut-derived cell line from the spruce budworm, Choristoneura fumiferana.

    PubMed

    Hu, Wenqi; Cook, Barbara J; Ampasala, Dinakara R; Zheng, Sichun; Caputo, Guido; Krell, Peter J; Retnakaran, Arthur; Arif, Basil M; Feng, Qili

    2004-02-01

    The morphological and molecular responses of a midgut-derived cell line of the spruce budworm, Choristoneura fumiferana, to 20-hydroxyecdysone (20E) and the nonsteroidal ecdysone agonist, tebufenozide (RH-5992), were investigated. The cells responded to these compounds by clumping, generating filamentous extensions, increased mortality and expression of the transcription factor, Choristoneura hormone receptor 3 (CHR3). This cell line can be used as a model system to study the mode of action of ecdysone and its agonists. With subsequent passaging in ecdysteroid-containing medium, the degree of clumping increased and the clumping could not be reversed by subculturing in ecdysteroid-free medium. Cell numbers of the adapted cell lines in 20E and RH-5992 containing media were not significantly decreased, compared to the control, but both cell lines accumulated less (14)C-labeled RH-5992 and lost the capability of expressing CHR3 in response to these compounds. Taken together, the cell lines appeared to develop a mechanism to adapt to the toxic effects of these compounds. Arch. Insect Biochem. Physiol. 55:68-78, 2004.

  7. Immunohistochemical localization of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in the superior olivary complex of mice after radiofrequency exposure.

    PubMed

    Maskey, Dhiraj; Kim, Myeung Ju

    2014-04-03

    Raising health concerns about the biological effects from radiofrequency exposure, even with conflicting results, has prompted calls for formulation of a guideline of the biological safety level. Given the close proximity between a mobile phone and the ear, it has been suggested that the central auditory system may be detrimentally influenced by radiofrequency exposure. In the auditory system, neurotrophins are important in the regulation of neuron survival, especially mammalian cochlear neurons. Neurotrophic factors like brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) present in the auditory system are responsible for the maintenance of auditory neurons. BDNF and GDNF may protect against acoustic trauma and prevent from hearing defect. The present study applied radiofrequency at a specific absorption rate (SAR) of 1.6W/kg (E1.6) or 0W/kg group to determine the distribution of BDNF and GDNF in the nuclei of superior olivary complex (SOC). In the E1.6 group, significant decrements of BDNF immunoreactivity (IR) were noted in the lateral superior olive, medial superior olive, superior paraolivary nucleus and medial nucleus of the trapezoid body. GDNF IR was also significantly decreased (p<0.001) in all SOC nuclei of the E1.6 group. The decrease in the IR of these neurotrophic factors in the SOC of the E1.6 group suggests a detrimental effect of RF exposure in the auditory nuclei.

  8. Inheritance of polyphenol oxidase activity in wheat breeding lines derived from matings of low polyphenol oxidase parents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenol oxidase (PPO) in grain plays a major role in time-dependent discoloration of wheat (Triticum aestivum L.) products, especially fresh noodles. Breeding wheat cultivars with low or nil PPO activity can reduce the undesirable product darkening. The low PPO line PI 117635 was crossed to two...

  9. Establishment and genetic characterization of ANGM-CSS, a novel, immortal cell line derived from a human glioblastoma multiforme.

    PubMed

    Notarangelo, Angelantonio; Trombetta, Domenico; D'Angelo, Vincenzo; Parrella, Paola; Palumbo, Orazio; Storlazzi, Clelia Tiziana; Impera, Luciana; Muscarella, Lucia Anna; La Torre, Antonella; Affuso, Andrea; Fazio, Vito Michele; Carella, Massimo; Zelante, Leopoldo

    2014-03-01

    Glioblastoma multiforme (World Health Organization, grade IV astrocytoma) is the most common and most aggressive malignant primary brain tumor. We report a novel cell line, designated as ANGM-CSS, which was established from a 56-year-old male patient with a surgically removed glioblastoma multiforme. The ANGM-CSS cell line was established in vitro and characterized using histological and immunohistochemical staining, classical and molecular cytogenetic analyses, molecular studies and functional assays using a xenograft model in immunodeficient animals. ANGM-CSS was positive for CD133, nestin and vimentin proteins, whereas GFAP showed staining only in a fraction of the cells. Cytogenetic and molecular cytogenetic analysis revealed a near-tetraploid karyotype, with a modal chromosome number from 88 to 91, and additional cytogenetic abnormalities, such as the t(6;14)(p12;q11.2), t(8;10)(q24.2;q21.1) and t(5;9)(q34;p21) unbalanced translocations. Moreover, ANGM-CSS showed amplification of the MET and EGFR genes whose overexpression was observed at the mRNA level. Interestingly, ANGM-CSS is tumorigenic when implanted in immunodeficient mice, and the cells obtained from the xenografts showed the same morphology and karyotype in vitro as the original cell line. ANGM-CSS represents a biologically relevant cell line to be used to investigate the molecular pathology of glioblastoma multiforme, also to evaluate the efficacy of novel therapeutic drugs in vitro.

  10. Nicotine, acetylcholine and bombesin are trophic growth factors in neuroendocrine cell lines derived from experimental hamster lung tumors

    SciTech Connect

    Schueller, H.M.; Nylen, E.; Park, P.; Becker, K.L. George Washington Univ., Washington, DC )

    1990-01-01

    Neuroendocrine hamster lung tumors, induced by exposure to 60% hyperoxia and subcutaneous administration of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) for 12 weeks, were placed in cell culture. By subsequent selective transfer of epithelial cells and maintenance in an atmosphere of 8% CO{sub 2}, cell lines with characteristics of neuroendocrine cells were established. The neuroendocrine markers expressed by these cell lines included electron dense neuroendocrine secretion granules as well as secretion of calcitonin and mammalian bombesin. In keeping with data previously reported for a human neuroendocrine lung tumor cell line, nicotine, acetylcholien, and mammalian bombesin (MB) acted as strongrowth factors in these neuroendocrine hamster tumor lines. The mitogenic effect of nicotine an acetylcholine was abolished by nicotinic receptor inhibition while the effects of mammalian bombesin were inhibited by an antagonist of MB receptors. Our data suggest that a receptor-mediated mitogenic effect of nicotine on neuroendocrine lung cells may be instrumental in the induction of smoking-associated small cell lung cancer.

  11. Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines.

    PubMed

    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.

  12. Hodgkin and sternberg-reed cell antigen(s) detected by an antiserum to a cell line (L428) derived from Hodgkin's disease.

    PubMed

    Stein, H; Gerdes, J; Kirchner, H; Schaadt, M; Diehl, V

    1981-10-15

    Antisera to the cell line L428, derived from Hodgkin's disease, were raised in rabbits by injecting L428 cells intravenously and subcutaneously. The anti-L428 cell serum that did not react with HLA-DR was absorbed with tonsil cell plus acute myeloid leukemia cells or tonsil cells plus neutrophils, monocytes, and blood lymphocytes. Then it was tested for its ability to discriminate between L428 cells, Hodgkin and Sternberg-Reed cells, and various other cells. It was found that the anti L428 cell serum absorbed with tonsil cells plus acute myeloid leukemia cells stained only L428 cells, Hodgkin and Sternberg-Reed cells, and neutrophils. The anti L428 cell serum absorbed with tonsil cell plus neutrophils, monocytes, and blood lymphocytes reacted with L428 cells and Hodgkin and sternberg-Reed cells from 13 cases of Hodgkin's disease. It did not react with any other cell type present in the blood or in lymphoid tissue or with cells from five cases of non-Hodgkin's lymphoma. The absorbed anti-L428 cell serum also failed to stain Daudi and HRIK cell line cells. We conclude that the anti-L428 cell serum defines an antigen that is apparently restricted in expression to L428 cells and Hodgkin and Sternberg-Reed cells. This is a strong indication that the L428 cell line cells are derived from Hodgkin and Sternberg-Reed cells.

  13. Vitamin D fails to prevent serum starvation- or staurosporine-induced apoptosis in human and rat osteosarcoma-derived cell lines

    SciTech Connect

    Witasp, Erika; Gustafsson, Ann-Catrin; Cotgreave, Ian; Lind, Monica . E-mail: monica.lind@imm.ki.se; Fadeel, Bengt . E-mail: bengt.fadeel@imm.ki.se

    2005-05-13

    Previous studies have suggested that 1,25(OH){sub 2}D{sub 3}, the active form of vitamin D{sub 3}, may increase the survival of bone-forming osteoblasts through an inhibition of apoptosis. On the other hand, vitamin D{sub 3} has also been shown to trigger apoptosis in human cancer cells, including osteosarcoma-derived cell lines. In the present study, we show that 1,25(OH){sub 2}D{sub 3} induces a time- and dose-dependent loss of cell viability in the rat osteosarcoma cell line, UMR-106, and the human osteosarcoma cell line, TE-85. We were unable, however, to detect nuclear condensation, phosphatidylserine externalization, or other typical signs of apoptosis in this model. Moreover, 1,25(OH){sub 2}D{sub 3} failed to protect against apoptosis induced by serum starvation or incubation with the protein kinase inhibitor, staurosporine. These in vitro findings are thus at variance with several previous reports in the literature and suggest that induction of or protection against apoptosis of bone-derived cells may not be a primary function of vitamin D{sub 3}.

  14. Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas

    USGS Publications Warehouse

    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.

  15. Evidence for epithelial-mesenchymal transition in cancer stem-like cells derived from carcinoma cell lines of the cervix uteri.

    PubMed

    Lin, Jiaying; Liu, Xishi; Ding, Ding

    2015-01-01

    The cancer stem cell (CSC) paradigm is one possible way to understand the genesis of cancer, and cervical cancer in particular. We quantified and enriched ALDH1(+) cells within cervical cancer cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). ALDH1 expression in spheroid-derived cells (SDC) and the parental monolayer-derived cell (MDC) line was compared by flow-cytometry. Invasion capability was evaluated by Matrigel assay and expression of EMT-related genes Twist 1, Twist 2, Snail 1, Snail 2, Vimentin and E-cadherin by real-time PCR. ALDH1 expression was significantly higher in SDC. ALDH1(+) cells showed increased colony-formation. SDC expressed lower levels of E-cadherin and elevated levels of Twist 1, Twist 2, Snail 1, Snail 2 and Vimentin compared to MDC. Cervical cancer cell lines harbor potential CSC, characterized by ALDH1 expression as well as properties like invasiveness, colony-forming ability, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

  16. Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

    PubMed Central

    Diaz Perez, Silvia V.; Kim, Rachel; Li, Ziwei; Marquez, Victor E.; Patel, Sanjeet; Plath, Kathrin; Clark, Amander T.

    2012-01-01

    Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1–UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. PMID:22058289

  17. 6-Aryl and heterocycle quinazoline derivatives as potent EGFR inhibitors with improved activity toward gefitinib-sensitive and -resistant tumor cell lines.

    PubMed

    Hamed, Mostafa M; Abou El Ella, Dalal A; Keeton, Adam B; Piazza, Gary A; Abadi, Ashraf H; Hartmann, Rolf W; Engel, Matthias

    2013-09-01

    A group of novel anilinoquinazoline derivatives with variable aryl and heterocyclic substituents at position 6 were synthesized and tested for their EGFR-inhibitory activity. Aryl and heterocyclic rings were attached to the quinazoline scaffold through different linkages such as imine, amide, and thiourea. Most of the aryl and heterocyclic derivatives showed potent inhibition of wild-type EGFR with IC₅₀ values in the low nanomolar range. Among these, thiourea derivatives 6 a, 6 b and compound 10 b also retained significant activity toward the gefitinib-insensitive EGFR(T790M/L858R) mutant, displaying up to 24-fold greater potency than gefitinib. In addition, cell growth inhibitory activity was tested against cancer cell lines with wild-type (KB cells) and mutant EGFR (H1975 cells). Several compounds including 6 a were found to be more potent than the reference compound gefitinib toward both cell lines, as was the case for compound 10 b against H1975 cells. Therefore, compounds 6 a and 10 b in particular may serve as new leads for the development of inhibitors effective against wild-type EGFR as well as gefitinib-resistant mutants.

  18. Apoptosis Activation in Human Lung Cancer Cell Lines by a Novel Synthetic Peptide Derived from Conus californicus Venom.

    PubMed

    Oroz-Parra, Irasema; Navarro, Mario; Cervantes-Luevano, Karla E; Álvarez-Delgado, Carolina; Salvesen, Guy; Sanchez-Campos, Liliana N; Licea-Navarro, Alexei F

    2016-02-05

    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.

  19. Apoptosis Activation in Human Lung Cancer Cell Lines by a Novel Synthetic Peptide Derived from Conus californicus Venom

    PubMed Central

    Oroz-Parra, Irasema; Navarro, Mario; Cervantes-Luevano, Karla E.; Álvarez-Delgado, Carolina; Salvesen, Guy; Sanchez-Campos, Liliana N.; Licea-Navarro, Alexei F.

    2016-01-01

    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. PMID:26861394

  20. SPECTROPHOTOMETRY OF THE HUYGENS REGION OF THE ORION NEBULA, THE EXTENDED ORION NEBULA, AND M 43: SCATTERED LIGHT SYSTEMATICALLY DISTORTS CONDITIONS DERIVED FROM EMISSION LINES

    SciTech Connect

    O'Dell, C. R.; Harris, Jessica A. E-mail: jessica.a.harris@vanderbilt.ed

    2010-10-15

    We report on medium resolution spectrophotometry of the Orion Nebula region, including for the first time the Extended Orion Nebula (EON) and the nearby M 43. The 49 long-slit observations were divided into 99 smaller samples, which have allowed determinations of the amount of extinction, extinction-corrected H{beta} surface brightness, electron temperatures (from [S II], [N II], and [O III]), and electron densities (from [S II] and [Cl III]) throughout much of this complex region. We verify an earlier conclusion from a radio/optical study that beyond about 5' from {theta}{sup 1}Ori C local emission begins to be contaminated by scattering of light from the much brighter central Huygens region of M 42, and this scattered light component becomes dominant at large distances. This contamination means that the derived properties for the outer regions are not accurate. From comparison of the light from the dominant star in M 43 with the continuum of that nebula (which is almost entirely scattered starlight), it is determined that scattered light is enhanced in the blue, which can lead to observed Balmer line ratios that are theoretically impossible and erroneous electron temperatures. This blue scattering of emission lines is important even in the Huygens region because it means that at anything except very high spectroscopic resolution the observed lines are a blend of the original and scattered light, with shorter wavelength lines being artificially enhanced. This can lead to overestimates of the electron temperatures derived from the nebular and auroral line ratios of forbidden lines. This phenomenon is probably applicable to many other H II regions. We have been able to use extinction-insensitive line ratios, the extinction-corrected surface brightness in H{beta}, and the equivalent width of the continuum to create for the first time a three-dimensional model of the entire M 42, EON, and M 43 region. This is an irregular concave blister of ionized gas bounded on the

  1. Evaluation of Exotically-Derived Soybean Breeding Lines for Seed Yield, Germination, Damage, and Composition under Dryland Production in the Midsouthern USA

    PubMed Central

    Bellaloui, Nacer; Smith, James R.; Mengistu, Alemu; Ray, Jeffery D.; Gillen, Anne M.

    2017-01-01

    Although the Early Soybean Production System (ESPS) in the Midsouthern USA increased seed yield under irrigated and non-irrigated conditions, heat stress and drought still lead to poor seed quality in heat sensitive soybean cultivars. Our breeding goal was to identify breeding lines that possess high germination, nutritional quality, and yield potential under high heat and dryland production conditions. Our hypothesis was that breeding lines derived from exotic germplasm might possess physiological and genetic traits allowing for higher seed germinability under high heat conditions. In a 2-year field experiment, breeding lines derived from exotic soybean accessions, previously selected for adaptability to the ESPS in maturity groups (MG) III and IV, were grown under non-irrigated conditions. Results showed that three exotic breeding lines had consistently superior germination across 2 years. These lines had a mean germination percentage of >80%. Two (25-1-1-4-1-1 and 34-3-1-2-4-1) out of the three lines with ≥80% germination in both years maintained high seed protein, oleic acid, N, P, K, B, Cu, and Mo in both years. Significant (P < 0.05) positive correlations were found between germination and oleic acid and with K and Cu in both years. Significant negative correlations were found between germination and linoleic acid, Ca, and hard seed in both years. There were positive correlations between germination and N, P, B, Mo, and palmitic acid only in 2013. A negative correlation was found between germination and green seed damage and linolenic acid in 2013 only. Seed wrinkling was significantly negatively correlated with germination in 2012 only. A lower content of Ca in the seed of high germinability genotypes may explain the lower rates of hard seed in those lines, which could lead to higher germination. Many of the differences in yield, germination, diseases, and seed composition between years are likely due to heat and rainfall differences between years. The

  2. Chemical Evolution of the Universe at 0.7 < z < 1.6 Derived from Abundance Diagnostics of the Broad-line Region of Quasars

    NASA Astrophysics Data System (ADS)

    Sameshima, H.; Yoshii, Y.; Kawara, K.

    2017-01-01

    We present an analysis of Mg ii λ2798 and Fe ii UV emission lines for archival Sloan Digital Sky Survey (SDSS) quasars to explore the diagnostics of the magnesium-to-iron abundance ratio in a broad-line region cloud. Our sample consists of 17,432 quasars selected from the SDSS Data Release 7 with a redshift range of 0.72 < z < 1.63. A strong anticorrelation between the Mg ii equivalent width (EW) and the Eddington ratio is found, while only a weak positive correlation is found between the Fe ii EW and the Eddington ratio. To investigate the origin of these differing behaviors of Mg ii and Fe ii emission lines, we perform photoionization calculations using the Cloudy code, where constraints from recent reverberation mapping studies are considered. We find from calculations that (1) Mg ii and Fe ii emission lines are created at different regions in a photoionized cloud, and (2) their EW correlations with the Eddington ratio can be explained by just changing the cloud gas density. These results indicate that the Mg ii/Fe ii flux ratio, which has been used as a first-order proxy for the Mg/Fe abundance ratio in chemical evolution studies with quasar emission lines, depends largely on the cloud gas density. By correcting this density dependence, we propose new diagnostics of the Mg/Fe abundance ratio for a broad-line region cloud. In comparing the derived Mg/Fe abundance ratios with chemical evolution models, we suggest that α-enrichment by mass loss from metal-poor intermediate-mass stars occurred at z ∼ 2 or earlier.

  3. Characterization of Two Novel Cell Lines with Distinct Heterogeneity Derived from a Single Human Bile Duct Carcinoma

    PubMed Central

    Zhang, Keqiang; Yu, Yong; Li, Bin; Li, Jiang; Yan, Zi; Hu, Zhenli; Yen, Yun; Wu, Mengchao; Jiang, Xiaoqing; Qian, Qijun

    2013-01-01

    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 of these two cell

  4. Establishment of hybridomas producing cancer specific human antibodies from B cell line derived from PBL of a patient with adult T cell leukemia.

    PubMed

    Kawahara, T; Ichikawa, A; Katakura, Y; Teruya, K; Yoshida, T; Kikuchi, M; Kamei, M; Hashizume, S; Shirahata, S

    2001-07-01

    Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of CD4(+) T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I) and concurs with an autoimmune disease and cancer due to attenuated immune response. In this study, we established ATL patient derived B-cell line TM-1 producing cancer-specific IgM antibodies, and further characterized its antigen specificity by establishing hybridomas fused with human-mouse origin hetero-myeloma cell line RF-S1. We established three hybridoma cell lines termed 2E12, 3E9, and 3E10, which continuously secreted human IgM antibodies. Immunohistochemical staining of formalin-fixed tissue section using antibodies secreted from these hybridomas showed that these antibodies specifically recognized tumor sites of human colon adenocarcinomas. Antibody produced from hybridoma 3E9 bound to some of leukemic cell lines, but not to normal human PBL, which was evidenced by the flow cytometric analysis, indicating that antibody produced from 3E9 recognizes cell surface antigen specifically expressed in the leukemic cells.

  5. A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo

    PubMed Central

    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

  6. Anthracene-9, 10-dione derivatives induced apoptosis in human cervical cancer cell line (CaSki) by interfering with HPV E6 expression.

    PubMed

    Sangthong, Supranee; Sangphech, Naunpun; Palaga, Tanapat; Ngamrojanavanich, Nattaya; Puthong, Songchan; Vilaivan, Tirayut; Muangsin, Nongnuj

    2014-04-22

    A new series of anthracene-9, 10-dione derivatives have been synthesized to increase cytotoxic activity against human papillomavirus (HPV) positive cancer cell line, CaSki. The highest cytotoxicity was achieved by 4-(benzylamino)-9,10-dioxo-4a,9,9a,10-tetrahydroanthracen-1-yl 4-ethylbenzenesulfonate (5) with the inhibitory concentration 50 (IC50) of 0.3 μM which is 20 times lower than that of cisplatin (CDDP; IC50 = 8.0 μM). The toxicity against non-cancerous cell line, WI-38, was low with the IC50 > 10 μM. Treatment with this compound resulted in decreasing HPV E6 expression. Furthermore, increasing p53 and decreasing Bcl-2 expression were noted. Cell cycle profiles revealed an accumulation of cells in the G2/M phase.

  7. Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

    PubMed Central

    Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

    2017-01-01

    Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

  8. Relationships between differential gene expression and heterosis in cotton hybrids developed from the foundation parent CRI-12 and its pedigree-derived lines.

    PubMed

    Zhu, Xinxia; Ainijiang; Zhang, Yuanming; Guo, Wangzen; Zhang, Tian-Zhen

    2011-02-01

    CRI-12, an Upland cotton variety with high yield, elite fiber quality and disease resistance, is further characterized by its high heritability, combining ability and genetic stability. CRI-12 and its pedigree-derived lines were used to develop increased heterosis cotton hybrids, including CRI-28, CRI-29, XZM 2 and Jimian18. CRI-12 was chosen as the cotton foundation parent and analyzed by gene differential expressions between hybrids and their corresponding parents at seedling, squaring and flowering stages. The following approaches were considered the most viable candidates to elucidate the molecular basis of CRI-12: cDNA-amplified fragment length polymorphisms (cDNA-AFLPs), gene differential expression ratios, and vegetative growth heterosis and yield heterosis for correlation analysis. The results indicated that CRI-12 plays a predominant role in vegetative heterosis in CRI-28, CRI-29 and Jimian18 hybrids at the seedling and squaring stages; the percentage of dominant expression in single parents was greater than other patterns; downregulated expression in single parents was higher than upregulated expression in hybrids, and downregulated expression in hybrids was the lowest of the four patterns in the three growth stage cumulative totals. The gene differential expression ratio of hybrids and parents varied for the three growth stages, suggesting gene differential expression changes over time. Further analysis of differential gene expression ratios, vegetative growth and yield heterosis correlation revealed upregulated expression in hybrids were correlated with vegetative heterosis at the seedling and squaring stages, which play an important role in yield heterosis at the flowering stage. Downregulated expression in the maternal parent (CRI-12 and its pedigree-derived lines) suggested benefits in vegetative heterosis at the squaring stage, but a possible hybrid yield decrease at the flowering stage. These results provided evidence that CRI-12 and its pedigree-derived

  9. Induction of apoptosis in the human promyelocytic leukemia cell line HL60 by falconensone A and its derivatives, new polyenes.

    PubMed

    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.

  10. TECHNICAL BASIS FOR DOE STANDARD 3013 EQUIVALENCY SUPPORTING REDUCED TEMPERATURE STABILIZATION OF OXALATE-DERIVED PLUTONIUM DIOXIDE PRODUCED BY THE HB-LINE FACILITY AT SAVANNAH RIVER SITE

    SciTech Connect

    Duffey, J. M.; Livingston, R. R.; Berg, J. M.; Veirs, D. K.

    2013-02-06

    This report documents the technical basis for determining that stabilizing highpurity PuO{sub 2} derived from oxalate precipitation at the SRS HB-Line facility at a minimum of 625 {degree}C for at least four hours in an oxidizing atmosphere is equivalent to stabilizing at a minimum of 950 {degree}C for at least two hours as regards meeting the objectives of stabilization defined by DOE-STD-3013 if the material is handled in a way to prevent excessive absorption of water.

  11. Complement-dependent antibody cytotoxicity test of chicken antibody with duck complement used against cells of a Marek's disease lymphoma-derived cell line (MSB-1).

    PubMed

    Sugimoto, C; Kodama, H; Mikami, T

    1979-01-01

    Complement-dependent antibody cytotoxicity (CDAC) against cells of a lymphoblastoid cell line (MSB-1) derived from Marek's disease lymphoma was investigated in a chicken antibody system using complements from several animal species. Cytotoxicity was seldom observed when rabbit, guinea pig, or chicken complements were used in the presence of hyperimmune chicken serum against MSB-1. With duck complement, however, cytotoxicity was always observed. Therefore, duck complement appears to be suitable for assay of hyperimmune chicken serum against MSB-1 cells by the CDAC test.

  12. Ukrain(TM), a semisynthetic Chelidonium majus alkaloid derivative, acts by inhibition of tubulin polymerization in normal and malignant cell lines.

    PubMed

    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).

  13. MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation.

    PubMed

    Stacchini, A; Aragno, M; Vallario, A; Alfarano, A; Circosta, P; Gottardi, D; Faldella, A; Rege-Cambrin, G; Thunberg, U; Nilsson, K; Caligaris-Cappio, F

    1999-02-01

    We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

  14. 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.

    PubMed

    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.

  15. Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as inhibitors of type 1 5α-reductase and on cancer cell line SK-LU-1.

    PubMed

    Silva-Ortiz, Aylin Viviana; Bratoeff, Eugene; Ramírez-Apan, Teresa; Heuze, Yvonne; Sánchez, Araceli; Soriano, Juan; Cabeza, Marisa

    2015-12-15

    Testosterone (T) plays a crucial role in prostate growth. In androgen-dependent tissues T is reduced to dihydrotestosterone (DHT) because of the presence of the 5α-reductase enzyme. This androgen is more active than T, since it has a higher affinity for the androgen receptor (AR). When this mechanism is altered, androgen-dependent diseases, including prostate cancer, could result. The aim of this study was to synthesize several 16-dehydropregnenolone acetate derivatives containing a triazole ring at C-21 and a linear or alicyclic ester moiety at C-3 of the steroidal skeleton. These steroids were designed as potential inhibitors of the activity of both types (1 and 2) of 5α-reductase. The cytotoxic activity of these compounds was also evaluated on a panel of PC-3, MCF7, and SK-LU-1 human cancer cell lines. The results from this study showed that with the exception of steroids 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-propionate and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-pentanoate, the compounds exhibit a lower inhibitory activity for both isoenzymes of 5α-reductase than finasteride. Furthermore the 3β-hydroxy-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-20-one and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-acetate derivatives display 80% cytotoxic activity on the SK-LU-1 cell line. These results also indicated that the triazole derivatives, which have a hydroxyl or acetoxy group at C-3, could have an anticancer effect, whereas the derivatives with a alicyclic ester group at C-3 do not show biological activity.

  16. Kisspeptin-10 Modulates the Proliferation and Differentiation of the Rhesus Monkey Derived Stem Cell Line: R366.4

    PubMed Central

    Huma, Tanzeel; Wang, Zhengbo; Rizak, Joshua; Ahmad, Fiaz; Shahab, Muhammad; Ma, YuanYe; Yang, Shangchuan; Hu, Xintian

    2013-01-01

    The rhesus monkey embryonic stem cell line R366.4 has been identified to differentiate into a number of cell types. However, it has not been well characterized for its response to drugs affecting reproductive endocrinology. Kisspeptins (KPs) are ligands for the GPR-54, which is known to modulate reproductive function. The current study was designed to determine the effect of the KP-10 peptide on R366.4 cells and to investigate the role of KP-GPR54 in the cell proliferation process. Four different doses (0.1, 1, 10, and 100 nM) of KP-10 and control were selected to evaluate cell growth parameters and cellular morphological changes over a 72 hr period. The cells were treated with kisspeptin-10 during the early rosette stage. Proliferation rates, analyzed by flow cytometry and cell count methods, were found to be decreased after treatment. Moreover, the number of rosettes was found to decrease following KP-10 treatments. Morphological changes consisting of neuronal projections were also witnessed. This suggested that KP-10 had an antiproliferative effect on R366.4 cells leading to a differentiation state and morphological changes consistent with neuronal stem cell development. The R366.4 stem cell line differentiates based on kisspeptin signaling and may be used to investigate reproductive cell endocrinology in vitro. PMID:24381507

  17. SSR marker and ITS cleaved amplified polymorphic sequence analysis of soybean x Glycine tomentella intersubgeneric derived lines.

    PubMed

    Zou, J J; Singh, R J; Hymowitz, T

    2004-08-01

    Wild perennial Glycine species are an invaluable gene resource for the cultivated soybean [ Glycine max (L.) Merr., 2 n=40]. However, these wild species have been largely unexplored in soybean breeding programs because of their extremely low crossability with soybean and the need to employ in vitro embryo rescue methods to produce F(1) hybrids. The objective of this study was to develop molecular markers to identify gene introgression from G. tomentella, a wild perennial Glycine species, to soybean. A selection of 96 soybean simple sequence repeat (SSR) markers was evaluated for cross-specific amplification and polymorphism in G. tomentella. Thirty-two SSR markers (33%) revealed specific alleles for G. tomentella PI 483218 (2 n=78). These SSR markers were further examined with an amphidiploid line (2 n=118) and monosomic alien addition lines (MAALs), each with 2 n=40 chromosomes from soybean and one from G. tomentella. The results show that the use of SSR markers is a rapid and reliable method to detect G. tomentella chromosomes in MAALs. We also developed a cleaved amplification polymorphism sequence (CAPS) marker according to the sequences of internal transcribed spacer (ITS) regions in soybean and G. tomentella. Four MAALs that carry the ITS (rDNA) locus from G. tomentella were identified. The SSR and ITS-CAPS markers will greatly facilitate the introgression and characterization of gene transfer from G. tomentella to soybean.

  18. Cytotoxic and apoptotic effects of leptocarpin, a plant-derived sesquiterpene lactone, on human cancer cell lines.

    PubMed

    Bosio, Claudia; Tomasoni, Giacomo; Martínez, Rolando; Olea, Andrés F; Carrasco, Héctor; Villena, Joan

    2015-12-05

    Sesquiterpene lactones have attracted much attention in drug research because they present a series of biological activities such as anticancer, antifungal, anti-inflammatory, antimicrobial and antioxidant. Leptocarpin (LTC) is a sesquiterpene lactone isolated from a native Chilean plant, Leptocarpha rivularis, which has been widely used in traditional medicine by Mapuche people. Previous work has demonstrated that LTC decreases cell viability of cancer cell lines. In this contribution, we analyze the mechanism of LTC cytotoxicity on different cancer cell lines. The results show that in all cases LTC induces an apoptotic process and inhibition of NF-κB. Apoptosis has been confirmed by observing condensation of chromatin, nuclear fragmentation, release of cytochrome c into the cytosol, and increasing of caspase-3 activity. It has also been found that LTC is an effective inhibitor of NF-κB, which suggests that leptocarpin-induced cytotoxicity involves in some degree the inhibition of NF-κB signaling pathway. The concentration at which LTC inhibits NF-κB activity to the control level is similar or even lower than that found for parthenolide and others sesquiterpene lactones. These results indicate that leptocarpine is a very interesting molecule that could be considered as therapeutic agent for cancer treatment.

  19. Homozygous deletion of the. alpha. - and. beta. sub 1 -interferon genes in human leukemia and derived cell lines

    SciTech Connect

    Diaz, M.O.; Ziemin, S.; Le Beau, M.M.; Pitha, P.; Smith, S.D.; Chilcote, R.R.; Rowley, J.D. )

    1988-07-01

    The loss of bands p21-22 from one chromosome 9 homologue as a consequence of a deletion of the short arm (del(9p)), unbalanced translocation, or monosomy 9 is frequently observed in the malignant cells of patients with lymphoid neoplasias, including acute lymphoblastic leukemia and non-Hodgkin lymphoma. The {alpha}- and {beta}{sub 1}-interferon genes have been assigned to this chromosome region (9p21-22). The authors now present evidence of the homozygous deletion of the interferon genes in neoplastic hematopoietic cell lines and primary leukemia cells in the presence or absence of chromosomal deletions that are detectable at the level of the light microscope. In these cell lines, the deletion of the interferon genes is accompanied by a deficiency of 5{prime}-methylthioadenosine phosphorylase, an enzyme of purine metabolism. These homozygous deletions may be associated with the loss of a tumor-suppressor gene that is involved in the development of these neoplasias. The relevant genes may be either the interferon genes themselves or a gene that has a tumor-suppressor function and is closely linked to them.

  20. Stress proteins and oxidative damage in a renal derived cell line exposed to inorganic mercury and lead.

    PubMed

    Stacchiotti, Alessandra; Morandini, Fausta; Bettoni, Francesca; Schena, Ilaria; Lavazza, Antonio; Grigolato, Pier Giovanni; Apostoli, Pietro; Rezzani, Rita; Aleo, Maria Francesca

    2009-10-29

    A close link between stress protein up-regulation and oxidative damage may provide a novel therapeutic tool to counteract nephrotoxicity induced by toxic metals in the human population, mainly in children, of industrialized countries. Here we analysed the time course of the expression of several heat shock proteins, glucose-regulated proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of inorganic mercury and lead. Concomitantly, we used morphological and biochemical methods to evaluate metal-induced cytotoxicity and oxidative damage. In particular, as biochemical indicators of oxidative stress we detected reactive oxygen species (ROS) and nitrogen species (RNS), total glutathione (GSH) and glutathione-S-transferase (GST) activity. Our results clearly demonstrated that mercury increases ROS and RNS levels and the expressions of Hsp25 and inducible Hsp72. These findings are corroborated by evident mitochondrial damage, apoptosis or necrosis. By contrast, lead is unable to up-regulate Hsp72 but enhances Grp78 and activates nuclear Hsp25 translocation. Furthermore, lead causes endoplasmic reticulum (ER) stress, vacuolation and nucleolar segregation. Lastly, both metals stimulate the over-expression of MTs, but with a different time course. In conclusion, in NRK-52E cell line the stress response is an early and metal-induced event that correlates well with the direct oxidative damage induced by mercury. Indeed, different chaperones are involved in the specific nephrotoxic mechanism of these environmental pollutants and work together for cell survival.

  1. Establishment and characterization of an oral mucosal melanoma cell line (MEMO) derived from a longstanding primary oral melanoma.

    PubMed

    Lourenço, Silvia V; Bologna, Sheyla B; Hsieh, Ricardo; Sangueza, Martin; Fernandes, Juliana D; Nico, Marcello M S

    2013-04-01

    Oral mucosal melanoma is rare. Its incidence peaks between 41 and 60 years of age; male/female ratio is 2:1. Preferred oral sites include hard palate and maxillary gingiva. Risk factors have not been clearly identified, but pigmented lesions may be present before the diagnosis of oral melanoma. We report an unusual case of oral mucosal melanoma of long-standing duration on hard palate and maxillary alveolar ridge in a male patient. Histopathologic features confirmed the diagnosis of invasive melanoma with a prominent in situ component. A cell lineage derived from the tumor was established and characterized, with phenotypic markers of melanocytes.

  2. Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots.

    PubMed

    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.

  3. Flash pyrolysis of coal, coal maceral, and coal-derived pyrite with on-line characterization of volatile sulfur compounds

    USGS Publications Warehouse

    Chou, I.-Ming; Lake, M.A.; Griffin, R.A.

    1988-01-01

    A Pyroprobe flash pyrolysis-gas chromatograph equipped with a flame photometric detector was used to study volatile sulfur compounds produced during the thermal decomposition of Illinois coal, coal macerals and coal-derived pyrite. Maximum evolution of volatile organic sulfur compounds from all coal samples occurred at a temperature of approximately 700??C. At this temperature, the evolution of thiophene, its alkyl isomers, and short-chain dialkyl sulfide compounds relative to the evolution of benzothiophene and dibenzothiophene compounds was greater from coal high in organic sulfur than from coal low in organic sulfur. The variation in the evolution of sulfur compounds observed for three separate coal macerals (exinite, vitrinite, and inertinite) was similar to that observed for whole coal samples. However, the variation trend for the macerals was much more pronounced. Decomposition of coal-derived pyrite with the evolution of elemental sulfur was detected at a temperature greater than 700??C. The results of this study indicated that the gas chromotographic profile of the volatile sulfur compounds produced during flash pyrolysis of coals and coal macerals varied as a function of the amount of organic sulfur that occurred in the samples. Characterization of these volatile sulfur compounds provides a better understanding of the behavior of sulfur in coal during the thermolysis process, which could be incorporated in the design for coal cleaning using flash pyrolysis techniques. ?? 1988.

  4. Diatom-Derived Polyunsaturated Aldehydes Activate Cell Death in Human Cancer Cell Lines but Not Normal Cells

    PubMed Central

    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. PMID:24992192

  5. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

    PubMed

    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.

  6. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    NASA Technical Reports Server (NTRS)

    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.

  7. Exosomes as Biomarker Enriched Microvesicles: Characterization of Exosomal Proteins Derived from a Panel of Prostate Cell Lines with Distinct AR Phenotypes

    PubMed Central

    Hosseini-Beheshti, Elham; Pham, Steven; Adomat, Hans; Li, Na; Tomlinson Guns, Emma S.

    2012-01-01

    Prostate cancer is the leading type of cancer diagnosed in men. In 2010, ∼217,730 new cases of prostate cancer were reported in the United States. Prompt diagnosis of the disease can substantially improve its clinical outcome. Improving capability for early detection, as well as developing new therapeutic targets in advanced disease are research priorities that will ultimately lead to better patient survival. Eukaryotic cells secrete proteins via distinct regulated mechanisms which are either ER/Golgi dependent or microvesicle mediated. The release of microvesicles has been shown to provide a novel mechanism for intercellular communication. Exosomes are nanometer sized cup-shaped membrane vesicles which are secreted from normal and cancerous cells. They are present in various biological fluids and are rich in characteristic proteins. Exosomes may thus have potential both in facilitating early diagnosis via less invasive procedures or be candidates for novel therapeutic approaches for castration resistance prostate cancer. Because exosomes have been shown previously to have a role in cell-cell communication in the local tumor microenvironment, conferring activation of numerous survival mechanisms, we characterized constitutive lipids, cholesterol and proteins from exosomes derived from six prostate cell lines and tracked their uptake in both cancerous and benign prostate cell lines respectively. Our comprehensive proteomic and lipidomic analysis of prostate derived exosomes could provide insight for future work on both biomarker and therapeutic targets for the treatment of prostate cancer. PMID:22723089

  8. A novel approach to evaluate the pharmacokinetic biocomparability of a monoclonal antibody derived from two different cell lines using simultaneous crossover design.

    PubMed

    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.

  9. Predominant role of plasma membrane monoamine transporters in monoamine transport in 1321N1, a human astrocytoma-derived cell line.

    PubMed

    Naganuma, Fumito; Yoshikawa, Takeo; Nakamura, Tadaho; Iida, Tomomitsu; Harada, Ryuichi; Mohsen, Attayeb S; Miura, Yamato; Yanai, Kazuhiko

    2014-05-01

    Monoamine neurotransmitters should be immediately removed from the synaptic cleft to avoid excessive neuronal activity. Recent studies have shown that astrocytes and neurons are involved in monoamine removal. However, the mechanism of monoamine transport by astrocytes is not entirely clear. We aimed to elucidate the transporters responsible for monoamine transport in 1321N1, a human astrocytoma-derived cell line. First, we confirmed that 1321N1 cells transported dopamine, serotonin, norepinephrine, and histamine in a time- and dose-dependent manner. Kinetics analysis suggested the involvement of low-affinity monoamine transporters, such as organic cation transporter (OCT) 2 and 3 and plasma membrane monoamine transporter (PMAT). Monoamine transport in 1321N1 cells was not Na(+) /Cl(-) dependent but was inhibited by decynium-22, an inhibitor of low-affinity monoamine transporters, which supported the importance of low-affinity transporters. RT-PCR assays revealed that 1321N1 cells expressed OCT3 and PMAT but no other neurotransmitter transporters. Another human astrocytoma-derived cell line, U251MG, and primary human astrocytes also exhibited the same gene expression pattern. Gene-knockdown assays revealed that 1321N1 and primary human astrocytes could transport monoamines predominantly through PMAT and partly through OCT3. These results might indicate that PMAT and OCT3 in human astrocytes are involved in monoamine clearance.

  10. Establishment and characterization of HROC69 - a Crohn´s related colonic carcinoma cell line and its matched patient-derived xenograft.

    PubMed

    Kuehn, Florian; Mullins, Christina S; Krohn, Mathias; Harnack, Christine; Ramer, Robert; Krämer, Oliver H; Klar, Ernst; Huehns, Maja; Linnebacher, Michael

    2016-04-18

    Colitis-associated colorectal cancer (CAC) seems to be a rather unique entity and differs in its genetic alterations, tumour formation capacities, and clinical features from sporadic colorectal carcinoma. Most descriptions about tumour biology of CAC refer to ulcerative colitis; data about Crohn´s colitis related carcinomas are scarce. The majority of patients with Crohn´s disease are under immunosuppression which generates a different environment for tumour growth. We first describe the clinical case of a fast growing CAC in a long-term immunosuppressed patient with Crohn´s disease and successful establishment and characterization of carcinoma cell lines along with their corresponding patient-derived xenograft. Subsequently, these tumor models were molecularly and functionally analysed. Beside numerous chromosomal alterations, mutations in TP53, APC, PTEN and SMAD3 were identified. The cell lines express numerous cancer testis antigens, surface molecules involved in immune evasion but low levels of HLA class I molecules. They show strong invasive but in comparison weak migratory activity. The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient. They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

  11. In Vitro Evaluation of the Antimicrobial Ability and Cytotoxicity on Two Melanoma Cell Lines of a Benzylamide Derivative of Maslinic Acid

    PubMed Central

    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

  12. Comment on "exact solutions of the derivative nonlinear Schrödinger equation for a nonlinear transmission line".

    PubMed

    Nickel, J; Schürmann, H W

    2007-03-01

    In a recent article Kengne and Liu [Phys. Rev. E 73, 026603 (2006)] have presented a number of exact elliptic solutions for a derivative nonlinear Schrödinger equation. It is the aim of this Comment to point out that all these solutions given in Secs. II and III of this article (referred to as KL in the following) are subcases of the general solution of Eq. (KL.9). Conditions for the parameters A-E of the solutions given by Kengne and Liu can be found from general conditions for solitary and periodic elliptic solutions as shown in the following. Positive and bounded solutions can be found by considering the phase diagram. Therefore, the comment of Kengne and Liu that "we find its particular positive bounded solutions" can be specified.

  13. Flavokawain derivative FLS induced G2/M arrest and apoptosis on breast cancer MCF-7 cell line

    PubMed Central

    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 1H-nuclear magnetic resonance, electron-impact mas spectrometry, infrared spectroscopy, and ultraviolet (1H 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. PMID:27358555

  14. 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.

    PubMed

    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 significantly reduced weight gain in all 6-hydroxydopamine-lesioned rats in this study. Following behavioural analysis where a confirmed improvement of behaviour was established, tissues were dissected for neurochemical analysis. In lesioned rats with intranigral injections of administered glial cell line-derived neurotrophic factor, significant increases of nigral, but not striatal tyrosine hydroxylase activity were measured. Additionally, 6-hydroxydopamine lesions significantly increased striatal dynorphin (61-139%) and met-enkephalin (81-139%), but not substance P levels. In these rats, intranigrally-administered glial cell line-derived neurotrophic factor injections reversed lesion-induced increases in nigral dynorphin A levels and increased nigral dopamine levels, but did not alter nigral met

  15. Synthesis and biological evaluation of some substituted-2-N-(5-chloro-2-methoxy-4-methylphenylsulphonyl) glutamic acid derivatives against prostate cancer cell line PC3.

    PubMed

    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.

  16. A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

    PubMed

    Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

    2017-02-09

    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.

  17. 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.

    PubMed

    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.

  18. Glial cell line-derived neurotrophic factor alters the growth characteristics and genomic imprinting of mouse multipotent adult germline stem cells

    SciTech Connect

    Jung, Yoon Hee

    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.

  19. Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV).

    PubMed

    Gong, J; Huang, Y; Huang, X; Ouyang, Z; Guo, M; Qin, Q

    2011-09-01

    A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.

  20. Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression.

    PubMed

    Ushmorov, Alexey; Ritz, Olga; Hummel, Michael; Leithäuser, Frank; Möller, Peter; Stein, Harald; Wirth, Thomas

    2004-11-15

    Immunoglobulin production is impaired in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) in spite of functional clonal rearrangements. The presence of "crippling" mutations in coding and regulatory regions, as well as down-regulation of B-cell-specific transcription factors, has been suggested as a potential reason for the lack of immunoglobulin (Ig) chain gene transcription. We have investigated the impact of epigenetic silencing in suppressing Ig heavy (H)-chain expression. Chromatin immunoprecipitation (ChIP) was used to analyze transcription factor binding to octamer motifs present in the IgH regulatory regions. Transcription factors were bound to these motifs in control cell lines, however, they were absent in the cHL-derived cell lines KMH2, L1236, and L428. Ectopic expression of octamer-binding transcription factor (Oct2) and/or B-cell Oct binding protein/Oct-binding factor (BOB.1/OBF.1) did not result in any measurable binding to these sites. Increased histone 3 Lysine 9 (H3-K9) methylation was observed in the promoter region of the IgH locus in L428 and L1236 cells. This is a typical feature of heterochromatic, transcriptionally silent regions. Treatment of cHL-derived cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) partially reactivated IgH transcription and affected chromatin modifications. Our results suggest an important role of epigenetic silencing in the inhibition of IgH transcription in HRS cells.

  1. Substance P enhances the proliferation and migration potential of murine bone marrow-derived mesenchymal stem cell-like cell lines.

    PubMed

    Dubon, Maria Jose; Park, Ki-Sook

    2015-04-01

    Due to the therapeutic characteristics of bone marrow (BM)-derived mesenchymal stem cells (MSCs), clinical trials are testing the use of autologous or allogeneic MSCs for the treatment of several conditions. These therapies require large numbers of MSCs and numerous studies are attempting to find substances that could enhance the egression of endogenous MSCs from the BM into the periphery and increase their proliferation in vivo and in vitro. It has been reported that substance P (SP) has the potential to increase the expansion of MSCs in vivo and to induce their mobilization from the BM into the periphery. The aim of the present study was to investigate the effects of SP on the migration and proliferation potential of two BM-derived MSC-like cell lines, ST2 and OP9. SP was found to induce the migration potential of ST2 cells in vitro. Furthermore, SP increased the proliferation of the MSCs cell line, OP9 cell line. Cyclin D1 expression was observed to increase in the OP9 cells, indicating the activation of the cell cycle in response to SP. The upstream signals involved in these phenomena have yet to be elucidated, although previous studies have proposed the activation of the extracellular signal-regulated kinase-1/2 and Wingless/β-catenin pathways as possible mediators of the cellular proliferation of human MSCs in response to SP. The present results therefore suggest that SP would facilitate the obtainment of higher numbers of endogenous MSCs from patients or donors and/or shorten the process of in vitro expansion that could cause the MSCs to undergo changes in their innate therapeutic characteristics prior to their use in therapy.

  2. Long-term mean and seasonal variation of altimetry-derived Oyashio transport across the A-line off the southeastern coast of Hokkaido, Japan

    NASA Astrophysics Data System (ADS)

    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.

  3. [Identification of a specific protein in flat revertant cell lines derived from ras oncogene-transformed cells].

    PubMed

    Fujita, H

    1990-03-01

    Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2 were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight X 10(-3) and pI) was detected only in the revertants, but not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. The polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts. Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. The p92-5.7 was not phosphorylated in the steady state of R1 cells. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. The expressions of gelsolin mRNA in the revertants were higher than in the EJ-NIH/3T3 cells. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of the specific protein expression in the flat revertants.

  4. Comparison of gene-specific DNA methylation patterns in equine induced pluripotent stem cell lines with cells derived from equine adult and fetal tissues.

    PubMed

    Hackett, Catherine H; Greve, Line; Novakofski, Kira D; Fortier, Lisa A

    2012-07-01

    Cellular pluripotency is associated with expression of the homeobox transcription factor genes NANOG, SOX2, and POU5F1 (OCT3/4 protein). Some reports suggest that mesenchymal progenitor cells (MPCs) may express increased quantities of these genes, creating the possibility that MPCs are more "pluripotent" than other adult cell types. The objective of this study was to determine whether equine bone marrow-derived MPCs had gene expression or DNA methylation patterns that differed from either early fetal-derived or terminally differentiated adult cells. Specifically, this study compared DNA methylation of the NANOG and SOX2 promoter regions and concurrent gene expression of NANOG, SOX2, and POU5F1 in equine induced pluripotent stem (iPS) cells, fetal fibroblasts, fetal brain cells, adult chondrocytes, and MPCs. Results indicate that NANOG and POU5F1 were not detectable in appreciable quantities in tissues other than the equine iPS cell lines. Equine iPS cells expressed large quantities of all three genes examined. Significantly increased quantities of SOX2 were noted in iPS cells and both fetal-derived cell types compared with adult cells. MPCs and adult chondrocytes expressed equivalent, low quantities of SOX2. Further, NANOG and SOX2 expression inversely correlated with the DNA methylation pattern in the promoter region, such that as gene expression increased, DNA methylation decreased. The equine iPS cell lines examined demonstrated DNA methylation and gene expression patterns that were consistent with pluripotency features described in other species. Results do not support previous reports that NANOG, SOX2, and POU5F1 are poised for increased activity in MPCs compared with other adult cells.

  5. Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies

    PubMed Central

    Devhare, Pradip B.; Desai, Swapnil; Lole, Kavita S.

    2016-01-01

    Hepatitis E virus (HEV) is a significant health problem in developing countries causing sporadic and epidemic forms of acute viral hepatitis. Hepatitis E is a self-limiting disease; however, chronic HEV infections are being reported in immunocompromised individuals. The disease severity is more during pregnancy with high mortality (20–25%), especially in third trimester. Early cellular responses after HEV infection are not completely understood. We analyzed innate immune responses associated with genotype-I HEV replication in human hepatoma cell lines (Huh7, Huh7.5 and HepG2/C3A) using HEV replicon system. These cells supported HEV replication with different efficiencies due to the cell type specific innate immune responses. HepG2/C3A cells were less supportive to HEV replication as compared to Huh7.5 and S10-3 cells. Reconstitution of the defective RIG-I and TLR3 signaling in Huh7.5 cells enabled them to induce higher level antiviral responses and restrict HEV replication, suggesting the involvement of both RIG-I and TLR3 in sensing HEV RNA and downstream activation of interferon regulatory factor 3 (IRF3) to generate antiviral responses. Inhibition of IRF3 mediated downstream responses in HepG2/C3A cells by pharmacological inhibitor BX795 significantly improved HEV replication efficiency implying the importance of this study in establishing a better cell culture system for future HEV studies. PMID:27230536

  6. Vasoactive Intestinal Peptide modulates trophoblast-derived cell line function and interaction with phagocytic cells through autocrine pathways

    PubMed Central

    Vota, Daiana; Paparini, Daniel; Hauk, Vanesa; Toro, Ayelén; Merech, Fatima; Varone, Cecilia; Ramhorst, Rosanna; Pérez Leirós, Claudia

    2016-01-01

    Trophoblast cells migrate and invade the decidual stroma in a tightly regulated process to maintain immune homeostasis at the maternal-placental interface during the first weeks of pregnancy. Locally synthesized factors modulate trophoblast cell function and their interaction with maternal leukocytes to promote the silent clearance of apoptotic cells. The vasoactive intestinal peptide (VIP) is a pleiotropic polypeptide with trophic and anti-inflammatory effects in murine pregnancy models. We explored the effect of VIP on two human first trimester trophoblast cell lines, particularly on their migration, invasiveness and interaction with phagocytic cells, and the signalling and regulatory pathways involved. We found that VIP enhanced trophoblast cell migration and invasion through the activation of high affinity VPAC receptors and PKA-CRE signalling pathways. VIP knocked-down trophoblast cells showed reduced migration in basal and leukemic inhibitor factor (LIF)-elicited conditions. In parallel, VIP-silenced trophoblast cells failed to induce the phagocytosis of apoptotic bodies and the expression of immunosuppressant markers by human monocytes. Our results suggest that VIP-mediated autocrine pathways regulate trophoblast cell function and contribute to immune homeostasis maintenance at placentation and may provide new clues for therapeutic intervention in pregnancies complicated by defective deep placentation. PMID:27212399

  7. Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue.

    PubMed

    Nobusue, Hiroyuki; Endo, Tsuyoshi; Kano, Koichiro

    2008-06-01

    We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro.

  8. Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

    PubMed

    Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

    2014-04-01

    The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

  9. Ultrastructural Characteristics of Three Undifferentiated Mouse Embryonic Stem Cell Lines and Their differentiated Three-Dimensional Derivatives: A Comparative Study

    PubMed Central

    Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah

    2014-01-01

    Abstract The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential. PMID:24606239

  10. Stability of multiple antigen receptor gene rearrangements and immunophenotype in Hodgkin's disease-derived cell line L428 and variant subline L428KSA.

    PubMed

    Athan, E S; Paietta, E; Papenhausen, P R; Augenlicht, L; Wiernik, P H; Gallagher, R E

    1989-07-01

    The Hodgkin's disease (HD) derived cell line L428 and a phorbol ester-selected subline L428KSA, which have been independently passaged in tissue culture for several years, were studied for possible antigen receptor gene and immunophenotypic differences. Multiple but identical alterations of these genes were found, including: the deletion of one and rearrangement of the other immunoglobulin (Ig) heavy chain allele; the rearrangement of one kappa and one lambda light chain allele; and the rearrangement of one T cell receptor (TCR) beta allele. Restriction mapping of the Ig heavy chain locus indicated that rearrangement of the retained allele produced a JH-C gamma 4 fusion product by an isotype switch mechanism. The 14q+ chromosome [t(14q32;?)] present in both cell cultures derived either from translocation 5' (telomeric) to the rearranged JH allele or 3' (centromeric) to the deleted Ig heavy chain allele and did not involve detectable rearrangement of the c-myc, bcl 1, or bcl 2 oncogenes. No differences in the immunophenotype were found between the L428 and L428KSA cells: both expressed leukocyte activation antigens and some determinants associated with myelomonocytic cells but no lymphoid markers. It is postulated that these phenotypic characteristics derived from secondary genetic events/differentiative reprogramming which produced extinction of primary lymphoid characters, including terminal deoxynucleotidyl transferase (TdT) essential to generation of the Ig and TCR gene rearrangements, and expression of an incomplete set of myelomonocytic markers.

  11. Sphere formation assay is not an effective method for cancer stem cell derivation and characterization from the Caco-2 colorectal cell line.

    PubMed

    Wu, Hui; Zhang, Haihong; Hu, Yue; Xia, Qiu; Liu, Chenlu; Li, Yingnan; Yu, Bin; Gu, Tiejun; Zhang, Xizhen; Yu, Xianghui; Kong, Wei

    2014-03-01

    Although the existence of cancer stem cells (CSCs) has been demonstrated in colorectal cancer, further investigation is hindered by controversies over their surface markers. The sphere formation assay is widely used as in vitro method for derivation and characterization of CSCs based on the intrinsic self-renewal property of these cells. Isolated cancer cells that form tumorspheres are generally recognized as CSCs with self-renewal and tumorigenic capacities. In this study, colon spheres grown from Caco-2 cells in the sphere formation assay were separated from other differentiated cells and characterized. Compared with Caco-2 cells, the derived colon spheres lost several CSC properties. The colon spheres contained decreased levels of specific colorectal CSC surface markers as well as low levels of ATP-binding cassette (ABC) transporters typically overexpressed in CSCs, resulting in the near loss of their chemoresistance ability. Furthermore, cells that developed as colon spheres with strong self-renewal ability in vitro lost their tumorigenic capacity in vivo compared with Caco-2 cells, which could establish tumors in non-obese diabetic/severe-combined immunodeficient (NOD/SCID) mice. The results indicated that the Caco-2 cell derived colon spheres did not consist of colorectal CSCs. Thus, the well-accepted sphere formation assay may not be an effective method for CSC isolation and characterization from the Caco-2 colorectal cancer cell line.

  12. Prenylated derivatives of baicalein and 3,7-dihydroxyflavone: synthesis and study of their effects on tumor cell lines growth, cell cycle and apoptosis.

    PubMed

    Neves, Marta Perro; Cidade, Honorina; Pinto, Madalena; Silva, Artur M S; Gales, Luís; Damas, Ana Margarida; Lima, Raquel T; Vasconcelos, M Helena; de São José Nascimento, Maria

    2011-06-01

    Fourteen baicalein and 3,7-dihydroxyflavone derivatives were synthesized and evaluated for their inhibitory activity against the in vitro growth of three human tumor cell lines. The synthetic approaches were based on the reaction with prenyl or geranyl bromide in alkaline medium, followed by cyclization of the respective monoprenylated derivative. Dihydropyranoflavonoids were also obtained by one-pot synthesis, using Montmorillonite K10 clay as catalyst combined with microwave irradiation. In vitro screening of the compounds for cell growth inhibitory activity revealed that the presence of one geranyl group was associated with a remarkable increase in the inhibitory activity. Moreover, for the 3,7-dihydroxyflavone derivatives a marked increase in growth inhibitory effect was also observed for compounds with furan and pyran fused rings. The most active compounds were also studied regarding their effect on cell cycle profile and induction of apoptosis. Overall the results point to the relevant role of the prenylation of flavone scaffold in the growth inhibitory activity of cancer cells.

  13. A novel spontaneous model of epithelial-mesenchymal transition (EMT) using a primary prostate cancer derived cell line demonstrating distinct stem-like characteristics.

    PubMed

    Harner-Foreman, Naomi; Vadakekolathu, Jayakumar; Laversin, Stéphanie A; Mathieu, Morgan G; Reeder, Stephen; Pockley, A Graham; Rees, Robert C; Boocock, David J

    2017-01-17

    Cells acquire the invasive and migratory properties necessary for the invasion-metastasis cascade and the establishment of aggressive, metastatic disease by reactivating a latent embryonic programme: epithelial-to-mesenchymal transition (EMT). Herein, we report the development of a new, spontaneous model of EMT which involves four phenotypically distinct clones derived from a primary tumour-derived human prostate cancer cell line (OPCT-1), and its use to explore relationships between EMT and the generation of cancer stem cells (CSCs) in prostate cancer. Expression of epithelial (E-cadherin) and mesenchymal markers (vimentin, fibronectin) revealed that two of the four clones were incapable of spontaneously activating EMT, whereas the others contained large populations of EMT-derived, vimentin-positive cells having spindle-like morphology. One of the two EMT-positive clones exhibited aggressive and stem cell-like characteristics, whereas the other was non-aggressive and showed no stem cell phenotype. One of the two EMT-negative clones exhibited aggressive stem cell-like properties, whereas the other was the least aggressive of all clones. These findings demonstrate the existence of distinct, aggressive CSC-like populations in prostate cancer, but, importantly, that not all cells having a potential for EMT exhibit stem cell-like properties. This unique model can be used to further interrogate the biology of EMT in prostate cancer.

  14. Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative

    SciTech Connect

    Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

    1982-08-01

    A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. The gaps may represent single-strand breaks. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or beta-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives.

  15. Resveratrol and some glucosyl, glucosylacyl, and glucuronide derivatives reduce Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes Scott A adhesion to colonic epithelial cell lines.

    PubMed

    Selma, María V; Larrosa, Mar; Beltrán, David; Lucas, Ricardo; Morales, Juan C; Tomás-Barberán, Francisco; Espín, Juan C

    2012-08-01

    The efficacy of resveratrol and some glucosyl, glucosylacyl, and glucuronide derivatives in inhibiting the adhesion of Salmonella Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes Scott A to Caco-2 and HT-29 colonic cells was investigated. The three bacteria strains were capable of adhering to both colonic epithelial cell lines, which responded by producing the pro-inflammatory interleukin 8 (IL-8). Adhesion inhibition of E. coli O157:H7 and S. Typhimurium to colonic cells was ≥60 and ≥40%, respectively, when resveratrol and most of the resveratrol derivatives were applied. Lower adhesion inhibition was observed for the bacteria with higher adherence potential, L. monocytogenes (≥20%). Resveratrol-3-O-(6'-O-butanoyl)-β-D-glucopyranoside (BUT) (50 and 100 μM) and resveratrol-3-O-(6'-O-octanoyl)-β-D-glucopyranoside (OCT) (50 μM) reduced IL-8 secretion by 100%. These results suggest that one mechanism for the beneficial attributes of resveratrol and especially the derivatives BUT and OCT could be the ability to reduce the adhesion and consequent pro-inflammatory cytokine production in intestinal epithelial cells in response to pathogen adhesion. The potential use of these compounds in the prevention of foodborne infections, intestinal homeostasis loss, and inflammatory bowel diseases could be another step in finding coadjuvants or alternatives to antibiotic treatments.

  16. A novel spontaneous model of epithelial-mesenchymal transition (EMT) using a primary prostate cancer derived cell line demonstrating distinct stem-like characteristics

    PubMed Central

    Harner-Foreman, Naomi; Vadakekolathu, Jayakumar; Laversin, Stéphanie A.; Mathieu, Morgan G.; Reeder, Stephen; Pockley, A. Graham; Rees, Robert C.; Boocock, David J.

    2017-01-01

    Cells acquire the invasive and migratory properties necessary for the invasion-metastasis cascade and the establishment of aggressive, metastatic disease by reactivating a latent embryonic programme: epithelial-to-mesenchymal transition (EMT). Herein, we report the development of a new, spontaneous model of EMT which involves four phenotypically distinct clones derived from a primary tumour-derived human prostate cancer cell line (OPCT-1), and its use to explore relationships between EMT and the generation of cancer stem cells (CSCs) in prostate cancer. Expression of epithelial (E-cadherin) and mesenchymal markers (vimentin, fibronectin) revealed that two of the four clones were incapable of spontaneously activating EMT, whereas the others contained large populations of EMT-derived, vimentin-positive cells having spindle-like morphology. One of the two EMT-positive clones exhibited aggressive and stem cell-like characteristics, whereas the other was non-aggressive and showed no stem cell phenotype. One of the two EMT-negative clones exhibited aggressive stem cell-like properties, whereas the other was the least aggressive of all clones. These findings demonstrate the existence of distinct, aggressive CSC-like populations in prostate cancer, but, importantly, that not all cells having a potential for EMT exhibit stem cell-like properties. This unique model can be used to further interrogate the biology of EMT in prostate cancer. PMID:28094783

  17. Accumulation of gamma-globin mRNA and induction of irreversible erythroid differentiation after treatment of CML cell line K562 with new doxorubicin derivatives.

    PubMed

    Szulawska, Agata; Arkusinska, Justyna; Czyz, Malgorzata

    2007-01-15

    Human chronic myelogenous leukemia (CML) cell line K562 can be chemically induced to differentiate and express embryonic and fetal globin genes. In this study, the effects of doxorubicin (DOX), an inducer of K562 cell erythroid differentiation, with those of epidoxorubicin (EDOX) as well as newly synthesized derivatives of both drugs (DOXM, DOXH, and EDOXM) on cell growth and differentiation were compared. Our results revealed that DOX, EDOX and their derivatives caused irreversible differentiation of K562 cells into more mature hemoglobin-containing cells. This phenomenon was linked to time-dependent inhibition of cell proliferation. Considering the impact of the structure of newly synthesized anthracyclines on their cellular activity, our data clearly indicated that among tested anthracyclines DOXM, a morpholine derivative of DOX exerted the highest antiproliferative and differentiating activity. An increase of gamma-globin mRNA level caused both by high transcription rate and by mRNA stabilization, as well as an enhancement of expression but not activity of erythroid transcription factor GATA-1 were observed. Therefore, a high level of hemoglobin-containing cells in the presence of DOXM resulted from transcriptional and post-transcriptional events on gamma-globin gene regulation. The same morpholine modification introduced to EDOX did not cause, however, similar effects on cellular level. Characterization of new powerful inducers of erythroid differentiation may contribute to the development of novel compounds for pharmacological approach by differentiation therapy to leukemia or to beta-globin disorder, beta-thalassemia.

  18. Phenotype versus immunoglobulin and T-cell receptor genotype of Hodgkin-derived cell lines: activation of immature lymphoid cells in Hodgkin's disease.

    PubMed

    Falk, M H; Tesch, H; Stein, H; Diehl, V; Jones, D B; Fonatsch, C; Bornkamm, G W

    1987-08-15

    Three Hodgkin-derived cell lines (L428, L540, and CO) were studied for rearrangements and expression of immunoglobulin and T-cell receptor genes, and their genotype was compared to the phenotype. As far as the genotype is concerned, all 3 cell lines have characteristics of lymphoid cells; L428 of B, and L540 and CO of T-cell origin. L428 cells have one Ig heavy chain allele rearranged to C gamma and transcribed into RNA, while the second is deleted. Furthermore, L428 cells show an unusual immunoglobulin kappa light chain gene rearrangement involving deletion of the kappa constant gene in one allele, while the remaining kappa and lambda loci are in germline configuration. L540 and CO have, in contrast to L428 cells, the immunoglobulin genes in germline and T-cell receptor genes rearranged. The T-cell receptor beta and gamma genes are rearranged in both L540 and CO, whereas a rearrangement in the alpha locus was detected in L540 cells only. RNA of the size of functional beta chain transcripts was found in CO cells and of the size of functional alpha chain transcripts in L540 cells. All 3 cell lines are classified as immature lymphoid cells with respect to the limited expression of B- and T-cell antigens, respectively, and to the incomplete expression of their antigen receptor. The immaturity of lymphoid differentiation contrasts with the expression of activation antigens, i.e. Ki-1, Ki-24, HLA-DR, and IL-2 receptor. The immaturity of the cells excludes the possibility that the cells were activated along the physiological pathway, i.e. by interaction of the cell with antigen. The results obtained on the cell lines are in accordance with in vivo studies and suggest that Hodgkin and Sternberg-Reed cells are immature lymphoid cells which are activated by a still unknown mechanism.

  19. High yield and rapid growth of Neoparamoeba pemaquidensis in co-culture with a rainbow trout gill-derived cell line RTgill-W1.

    PubMed

    Lee, L E J; Van Es, S J; Walsh, S K; Rainnie, D J; Donay, N; Summerfield, R; Cawthorn, R J

    2006-08-01

    Neoparamoeba pemaquidensis is an ubiquitous amphizoic marine protozoan and has been implicated as the causative agent for several diseases in marine organisms, most notably amoebic gill disease (AGD) in Atlantic salmon. Despite several reports on the pathology of AGD, relatively little is known about the protozoan and its relationship to host cells. In this study, an in vitro approach using monolayers of a rainbow trout gill cell line (RTgill-W1, ATCC CRL-2523) was used to rapidly grow large numbers of N. pemaquidensis (ATCC 50172) and investigate cell-pathogen interactions. Established cell lines derived from other tissues of rainbow trout and other fish species were also evaluated for amoeba growth support. The amoebae showed preference and highest yield when grown with RTgill-W1 over nine other tested fish cell lines. Amoeba yields could reach as high as 5 x 10(5) cells mL(-1) within 3 days of growth on the gill cell monolayers. The amoebae caused visible focal lesions in RTgill-W1 monolayers within 24 h of exposure and rapidly proliferated and spread with cytopathic effects destroying the neighbouring pavement-like cells within 48-72 h after initial exposure in media above 700 mOsm kg(-1). Disruption of the integrity of the gill cell monolayers could be noted within 30 min of exposure to the amoeba suspensions by changes in transepithelial resistance (TER) compared with control cell monolayers maintained in the exposure media. This was significantly different by 2 h (P < 0.05) compared with control cells and remained significantly different (P < 0.01) for the remaining 72 h that the TER was monitored. The RTgill-W1 cell line is thus a convenient model for growing N. pemaquidensis and for studying host-pathogen interactions in AGD.

  20. Isolation of Hokkaido virus, genus Hantavirus, using a newly established cell line derived from the kidney of the grey red-backed vole (Myodes rufocanus bedfordiae).

    PubMed

    Sanada, Takahiro; Seto, Takahiro; Ozaki, Yuka; Saasa, Ngonda; Yoshimatsu, Kumiko; Arikawa, Jiro; Yoshii, Kentaro; Kariwa, Hiroaki

    2012-10-01

    Hantaviruses belong to the family Bunyaviridae and are maintained in wild rodents. Although Vero E6 cells, which originate from African green monkey kidney, are used widely in hantavirus research, isolation of hantaviruses from this cell line is difficult. To develop an efficient method of propagation and isolation of hantaviruses we established a novel cell line, MRK101, derived from the kidney of the grey red-backed vole (Myodes rufocanus bedfordiae), the natural host of Hokkaido virus (HOKV). The MRK101 cells showed a significantly higher susceptibility to Puumala virus (PUUV) hosted by Myodes glareolus than Vero E6 cells. Viral nucleocapsid protein in PUUV-infected MRK101 cells was detected earlier than in Vero E6 cells, and the viral titre in the culture fluid of MRK101 cells was higher than that of Vero E6 cells during the early phase of infection. In contrast, MRK101 cells showed no susceptibility to Hantaan virus. HOKV, which has not been isolated to date, was isolated successfully using MRK101 cells. Moreover, the newly isolated HOKV was successfully propagated in MRK101, but not Vero E6, cells. Phylogenic analyses of the S (small), M (medium) and L (large) segment sequences revealed that HOKV is related most closely to PUUV, but is distinct from other hantaviruses. These data suggest that the MRK101 cell line is a useful tool for the isolation and propagation of hantaviruses. Moreover, this is (to our knowledge) the first report of hantavirus isolation in a cell line that originated from the natural host.

  1. Mast cell growth-enhancing activity (MEA) stimulates interleukin 6 production in a mouse bone marrow-derived mast cell line and a malignant subline.

    PubMed

    Hültner, L; Moeller, J

    1990-09-01

    A novel mast cell growth-enhancing activity (MEA/P40/interleukin 9 [IL-9]) purified from the conditioned medium of a murine interleukin 2 (IL-2)-dependent Mlsa-specific T-cell line (MLS4.2) was tested for its capacity to induce interleukin 6 (IL-6) production in a mouse bone marrow-derived factor-dependent mast cell line (L138.8A). This interleukin 3 (IL-3)/interleukin 4 (IL-4)/MEA-responsive cell line was demonstrated recently to express IL-6 mRNA and to secrete IL-6 when cultured with IL-3/IL-4. Now we were able to show that conditioned medium from L138.8A mast cells stimulated with MEA alone contained growth factor activity for the IL-6-dependent mouse hybridoma cell line 7TD1 that was completely blocked by the monoclonal anti-IL-6 antibody 6B4. A dose-response study including IL-3, IL-4, and MEA tested either alone or in different combinations revealed that among these growth factors MEA was the most potent inducer of IL-6 in L138.8A cells. Moreover, IL-4 but not IL-3 had a strong synergistic effect on MEA-induced IL-6 production. The autonomous malignant mast cell subline L138Cauto also showed enhanced IL-6 production when stimulated with MEA. Our findings indicate that MEA (IL-9) not only provides a proliferation signal, but also leads to a marked functional activation of responsive mast cells.

  2. Pigment Epithelium-Derived Factor (PEDF) Protects Osteoblastic Cell Line from Glucocorticoid-Induced Apoptosis via PEDF-R

    PubMed Central

    Yao, Shengcheng; Zhang, Yingnan; Wang, Xiaoyu; Zhao, Fengchao; Sun, Maji; Zheng, Xin; Dong, Hongyan; Guo, Kaijin

    2016-01-01

    Pigment epithelial-derived factor (PEDF) is known as a widely expressed multifunctional secreted glycoprotein whose biological actions are cell-type dependent. Recent studies demonstrated that PEDF displays cytoprotective activity in several cell types. However, it remains unknown whether PEDF is involved in glucocorticoid-induced osteoblast death. The aim of this study was to examine the role of PEDF in osteoblast survival in response to dexamethasone, an active glucocorticoid analogue, and explore the underlying mechanism. In the present study, dexamethasone (DEX) was used to induce MC3T3-E1 pre-osteoblast apoptosis. PEDF mRNA and protein levels and cell apoptosis were determined respectively. Then PEDF receptor (PEDF-R)- and lysophosphatidic acid (LPA)-related signal transductions were assessed. Here we show that DEX down-regulates PEDF expression, which contributes to osteoblast apoptosis. As a result, exogenous recombinant PEDF (rPEDF) inhibited DEX-induced cell apoptosis. We confirmed that PEDF-R was expressed on MC3T3-E1 pre-osteoblast membrane and could bind to PEDF which increased the level of LPA and activated the phosphorylation of Akt. Our results suggest that PEDF attenuated DEX-induced apoptosis in MC3T3-E1 pre-osteoblasts through LPA-dependent Akt activation via PEDF-R. PMID:27187377

  3. Genetically modified Schwann cells producing glial cell line-derived neurotrophic factor inhibit neuronal apoptosis in rat spinal cord injury.

    PubMed

    Liu, Guomin; Wang, Xukai; Shao, Guoxi; Liu, Qinyi

    2014-04-01

    Schwann cells (SCs) are the major cells constituting the peripheral nerve structure and function, and also secret a variety of neurotrophic factors. Schwann cell (SC) transplantation has recently emerged as a promising therapeutic strategy for spinal cord injury (SCI). In the present study, the ability of genetically modified SCs producing high levels of glial cell line‑derived neurotrophic factor (GDNF) to promote spinal cord repair was assessed. The GDNF gene was transduced into SCs. The engineered SCs were characterized by their ability to express and secrete biologically active GDNF, which was shown to inhibit apoptosis of primary rat neurons induced by radiation, and upregulate the expression of B‑cell lymphoma 2 (Bcl‑2) and downregulate the expression of Bcl‑2 associated X protein (Bax) in vitro. Following SC implantation into the spinal cord of adult rats with SCI induced by weight‑drop impact, the survival of rats with transplanted SCs, histology of the spinal cord and expression levels of Bcl‑2 and Bax were examined. Transplantation of unmodified and genetically modified SCs producing GDNF attenuated SCI by inhibiting apoptosis via the Bcl‑2/Bax pathways. The genetically modified SCs demonstrated markedly improved recovery of SCI as compared with unmodified SCs. The present study combined the outgrowth‑promoting property of SCs with the neuroprotective effects of overexpressed GDNF and identified this as a potential novel therapeutic strategy for SCI.

  4. Genotoxic and chemopreventive assessment of Cynara scolymus L. aqueous extract in a human-derived liver cell line.

    PubMed

    da Silva, Regiane Pereira; Jacociunas, Laura Vicedo; de Carli, Raíne Fogliati; de Abreu, Bianca Regina Ribas; Lehmann, Mauricio; da Silva, Juliana; Ferraz, Alexandre de Barros Falcão; Dihl, Rafael Rodrigues

    2017-02-01

    Cynara scolymus L., popularly known as artichoke, is consumed as food and used as tea infusions for pharmacological purposes to treat liver dysfunctions and other conditions. Scientific data on the safety and protective effect of artichoke in human-derived liver cells is missing. This study investigated the genotoxic and modulatory effect of a liophilized extract suspended in water of C. scolymus L. leaves. Four extract concentrations (0.62, 1.25, 2.5 and 5.0 mg/mL) were evaluated using the comet assay on human hepatocyte cultures, HepG2 cells. Genotoxicity was assessed after two treatment periods, 1 and 24 h. Antigenotoxicity was evaluated against oxidative lesions induced by hydrogen peroxide in pre-, simultaneous and post-treatment protocols. Artichoke leaves aqueous extract induced genotoxic effects in HepG2 cells after 1- and 24-h treatments. In turn, extract concentrations of 0.62, 1.25 and 2.5 mg/mL, exhibited a protective effect in pretreatment, compared to hydrogen peroxide alone. However, in simultaneous and post-treatment protocols, only the lowest concentration reduced the frequency of DNA damage induced by hydrogen peroxide. In addition, in the simultaneous treatment protocol, the highest artichoke extract concentration increased hydrogen peroxide genotoxicity. It can be concluded that artichoke is genotoxic, in vitro, to HepG2 cells, but can also modulate hydrogen peroxide DNA damage.

  5. SVCT-2 determines the sensitivity to ascorbate-induced cell death in cholangiocarcinoma cell lines and patient derived xenografts.

    PubMed

    Wang, Changzheng; Lv, Hongwei; Yang, Wen; Li, Ting; Fang, Tian; Lv, Guishuai; Han, Qin; Dong, Liwei; Jiang, Tianyi; Jiang, Beige; Yang, Guangshun; Wang, Hongyang

    2017-04-04

    Cholangiocarcinoma (CC) is a devastating malignancy with late diagnosis and poor response to conventional chemotherapy. Recent studies have revealed anti-cancer effect of vitamin C (l-ascorbic acid, ascorbate) in several types of cancer. However, the effect of l-ascorbic acid (AA) in CC remains elusive. Herein, we demonstrated that AA induced cytotoxicity in CC cells by generating intracellular reactive oxygen species (ROS), and subsequently DNA damage, ATP depletion, mTOR pathway inhibition. Moreover, AA worked synergistically with chemotherapeutic agent cisplatin to impair CC cells growth both in vitro and in vivo. Intriguingly, sodium-dependent vitamin C transporter 2 (SVCT-2) expression was inversely correlated with IC50 values of AA. Knockdown of SVCT-2 dramatically alleviated DNA damage, ATP depletion, and inhibition of mTOR pathway induced by AA. Furthermore, SVCT-2 knockdown endowed CC cells with the resistance to AA treatment. Finally, the inhibitory effects of AA were further confirmed in patient-derived CC xenograft models. Thus, our results unravel therapeutic potential of AA alone or in combination with cisplatin for CC. SVCT2 expression level may serve as a positive outcome predictor for AA treatment in CC.

  6. Pigment Epithelium-Derived Factor (PEDF) Protects Osteoblastic Cell Line from Glucocorticoid-Induced Apoptosis via PEDF-R.

    PubMed

    Yao, Shengcheng; Zhang, Yingnan; Wang, Xiaoyu; Zhao, Fengchao; Sun, Maji; Zheng, Xin; Dong, Hongyan; Guo, Kaijin

    2016-05-13

    Pigment epithelial-derived factor (PEDF) is known as a widely expressed multifunctional secreted glycoprotein whose biological actions are cell-type dependent. Recent studies demonstrated that PEDF displays cytoprotective activity in several cell types. However, it remains unknown whether PEDF is involved in glucocorticoid-induced osteoblast death. The aim of this study was to examine the role of PEDF in osteoblast survival in response to dexamethasone, an active glucocorticoid analogue, and explore the underlying mechanism. In the present study, dexamethasone (DEX) was used to induce MC3T3-E1 pre-osteoblast apoptosis. PEDF mRNA and protein levels and cell apoptosis were determined respectively. Then PEDF receptor (PEDF-R)- and lysophosphatidic acid (LPA)-related signal transductions were assessed. Here we show that DEX down-regulates PEDF expression, which contributes to osteoblast apoptosis. As a result, exogenous recombinant PEDF (rPEDF) inhibited DEX-induced cell apoptosis. We confirmed that PEDF-R was expressed on MC3T3-E1 pre-osteoblast membrane and could bind to PEDF which increased the level of LPA and activated the phosphorylation of Akt. Our results suggest that PEDF attenuated DEX-induced apoptosis in MC3T3-E1 pre-osteoblasts through LPA-dependent Akt activation via PEDF-R.

  7. Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.

    PubMed

    Böcker, Wolfgang; Yin, Zhanhai; Drosse, Inga; Haasters, Florian; Rossmann, Oliver; Wierer, Matthias; Popov, Cvetan; Locher, Melanie; Mutschler, Wolf; Docheva, Denitsa; Schieker, Matthias

    2008-08-01

    Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

  8. NTRK1 rearrangement in colorectal cancer patients: evidence for actionable target using patient-derived tumor cell line

    PubMed Central

    Hong, Min Eui; Jang, Jiryeon; Yoon, Nara; Ahn, Soo Min; Murphy, Danielle; Christiansen, Jason; Wei, Ge; Hornby, Zachary; Lee, Dong Woo; Park, Joon Oh; Park, Young Suk; Lim, Ho Yeong; Hong, Sung No; Kim, Seok-Hyeong; Kang, Won Ki; Park, Keunchil; Park, Woong Yang; Kim, Kyoung-Mee; Lee, Jeeyun

    2015-01-01

    Background We have investigated the incidence of NTRK1 rearrangements in metastatic gastrointestinal cancer patients and demonstrated the potential for clinical response of these patients to targeted therapy. Methods We prospectively collected tumor tissue specimens for one year and simultaneously generated patient-derived tumor cells (PDCs). Specimens were initially screened for TrkA protein expression using TrkA immunohistochemistry (IHC). In the case of TrkA IHC positive results, samples were further examined by fluorescence in situ hybridization (FISH) and next generation sequencing (NGS) to confirm the presence of NTRK1 rearrangements. Results From January 2014 to December 2014, a total of 74 metastatic colorectal cancer (CRC) patients and 66 gastric cancer (GC) patients were initially screened by TrkA IHC. Two of the 74 CRC patients (2.7%) and one of the 66 GC patients (1.5%) were positive for TrkA expression by IHC. All three IHC positive cases had evidence of NTRK1 rearrangements by FISH. NGS was performed on the 3 IHC positive cases and confirmed TPM3-NTRK1 rearrangements in the two CRC cases. One GC patient with TrkA expression by IHC did not harbor an NTRK1 rearrangement. PDCs established from the NTRK1 positive CRC patients were positive for the NTRK1 rearrangement. Entrectinib, a pan-TRK inhibitor, profoundly inhibited cell proliferation of NTRK1-rearranged PDCs with such inhibition associated with inactivation of TrkA, and down-regulation of downstream signaling pathways. Conclusion TrkA IHC is an effective, initial screening method for NTRK1 rearrangement detection in the clinic. Inhibition of the TrkA kinase is a promising targeted therapy for cancer patients whose tumors harbor a NTRK1 rearrangement. PMID:26472021

  9. Molecular and Cellular Characterization of a Zebrafish Optic Pathway Tumor Line Implicates Glia-Derived Progenitors in Tumorigenesis

    PubMed Central

    Solin, Staci L.; Wang, Ying; Mauldin, Joshua; Schultz, Laura E.; Lincow, Deborah E.; Brodskiy, Pavel A.; Jones, Crystal A.; Syrkin-Nikolau, Judith; Linn, Jasmine M.; Essner, Jeffrey J.; Hostetter, Jesse M.; Whitley, Elizabeth M.; Cameron, J. Douglas; Chou, Hui-Hsien; Severin, Andrew J.; Sakaguchi, Donald S.; McGrail, Maura

    2014-01-01

    In this study we describe the molecular and cellular characterization of a zebrafish mutant that develops tumors in the optic pathway. Heterozygous Tg(flk1:RFP)is18 transgenic adults develop tumors of the retina, optic nerve and optic tract. Molecular and genetic mapping demonstrate the tumor phenotype is linked to a high copy number transgene array integrated in the lincRNA gene lincRNAis18/Zv9_00007276 on chromosome 3. TALENs were used to isolate a 147kb deletion allele that removes exons 2–5 of the lincRNAis18 gene. Deletion allele homozygotes are viable and do not develop tumors, indicating loss of function of the lincRNAis18 locus is not the trigger for tumor onset. Optic pathway tumors in the Tg(flk1:RFP)is18 mutant occur with a penetrance of 80–100% by 1 year of age. The retinal tumors are highly vascularized and composed of rosettes of various sizes embedded in a fibrous matrix. Immunohistochemical analysis showed increased expression of the glial markers GFAP and BLBP throughout retinal tumors and in dysplastic optic nerve. We performed transcriptome analysis of pre-tumorous retina and retinal tumor tissue and found changes in gene expression signatures of radial glia and astrocytes (slc1a3), activated glia (atf3, blbp, apoeb), proliferating neural progenitors (foxd3, nestin, cdh2, her9/hes1), and glioma markers (S100β, vim). The transcriptome also revealed activation of cAMP, Stat3 and Wnt signal transduction pathways. qRT-PCR confirmed >10-fold overexpression of the Wnt pathway components hbegfa, ascl1a, and insm1a. Together the data indicate Müller glia and/or astrocyte-derived progenitors could contribute to the zebrafish Tg(flk1:RFP)is18 optic pathway tumors. PMID:25485542

  10. Derivation of Instantaneous Wye and Zero-Phase Sequence Voltages from Line-Line Voltages in Unbalanced 3-Phase 3-Wire Systems and Application of This Method to 3-Phase PWM Converter Control

    NASA Astrophysics Data System (ADS)

    Yuzurihara, Itsuo; Kawamura, Atsuo

    In general, voltage imbalances in 3-phase AC power systems are inevitable. 3-Phase PWM (Pulse Width Modulation) converter used in 3-wire systems are generally designed for use under limited imbalances of input voltages, and problems such as input current distortion, deterioration of output properties, degradation of efficiency and failure may occur in some cases. These problems cause severe damages to industries in some cases, for example, semiconductor production machines: SEMI defined “SEMI F47-0200” and “SEMI F47-0706” standards that have to be satisfied to realize voltage sag immunity. In order to compensate the remained problems due to the unbalanced input voltages, particular storage devices are designed additionally for conventional converters. This paper proposes that the determination of both the instantaneous zero-phase sequence voltage and wye voltages is essential for 3-phase PWM converter control used for a 3-wire system to keep its output rated under occasional or long-term voltage imbalances in an AC system. This paper also describes a general new method to derive the components of the voltages of instantaneous wye and zero-phase sequence voltage from line-line voltages of a 3-wire system. This paper also describes a method to apply the voltages to control the converter. The results obtained on implementation verify that this new converter keeps its output rated under unbalanced conditions wider than those defined by SEMIs without particular storage devices as far as the AC voltages are remained live.

  11. E-cadherin-downregulation and RECK-upregulation are coupled in the non-malignant epithelial cell line MCF10A but not in multiple carcinoma-derived cell lines.

    PubMed

    Yuki, Kanako; Yoshida, Yoko; Inagaki, Ryosaku; Hiai, Hiroshi; Noda, Makoto

    2014-04-02

    Expression of a mesenchymal phenotype is often associated with invasive/metastatic behaviors of carcinoma cells. Acquisition of a mesenchymal phenotype by a carcinoma cell is known as epithelial-mesenchymal transition (EMT). The membrane-anchored matrix metalloproteinase-regulator RECK is abundant in normal mesenchymal cells. In aggressive carcinomas, however, RECK expression is often downregulated. This apparent paradox prompted us to clarify the relationship between EMT and RECK. We found that TGFβ-induced E-cadherin downregulation, a hallmark of EMT, is accompanied by RECK-upregulation in a non-tumorigenic epithelial cell line (MCF10A). In contrast, the loss of E-cadherin expression is uncoupled from RECK-upregulation in carcinoma-derived cell lines (MCF7, MDA-MB-231, and A549). When RECK was artificially expressed in A549 cells, it showed little effect on EMT but elevated the level of integrin α5 and attenuated cell proliferation and migration. These findings implicate RECK in the regulation of proliferation and migration of normal epithelial cells after EMT and suggest how the uncoupling between EMT and RECK-upregulation impacts on the fates and behaviors of carcinoma cells.

  12. Development and persistence of kindling epilepsy are impaired in mice lacking glial cell line-derived neurotrophic factor family receptor α2

    PubMed Central

    Nanobashvili, Avtandil; Airaksinen, Matti S.; Kokaia, Merab; Rossi, Jari; Asztély, Fredrik; Olofsdotter, Klara; Mohapel, Paul; Saarma, Mart; Lindvall, Olle; Kokaia, Zaal

    2000-01-01

    Seizure activity regulates gene expression for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and their receptor components, the transmembrane c-Ret tyrosine kinase and the glycosylphosphatidylinositol-anchored GDNF family receptor (GFR) α1 and α2 in limbic structures. We demonstrate here that epileptogenesis, as assessed in the hippocampal kindling model, is markedly suppressed in mice lacking GFRα2. Moreover, at 6 to 8 wk after having reached the epileptic state, the hyperexcitability is lower in GFRα2 knock-out mice as compared with wild-type mice. These results provide evidence that signaling through GFRα2 is involved in mechanisms regulating the development and persistence of kindling epilepsy. Our data suggest that GDNF and NRTN may modulate seizure susceptibility by altering the function of hilar neuropeptide Y-containing interneurons and entorhinal cortical afferents at dentate granule cell synapses. PMID:11050250

  13. Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein.

    PubMed Central

    Hortin, G; Green, E D; Baenziger, J U; Strauss, A W

    1986-01-01

    Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides. Images Fig. 1. Fig. 2. Fig. 3. PMID:3017304

  14. Structural optimization of diphenylpyrimidine derivatives (DPPYs) as potent Bruton's tyrosine kinase (BTK) inhibitors with improved activity toward B leukemia cell lines.

    PubMed

    Zhao, Dan; Huang, Shanshan; Qu, Menghua; Wang, Changyuan; Liu, Zhihao; Li, Zhen; Peng, Jinyong; Liu, Kexin; Li, Yanxia; Ma, Xiaodong; Shu, Xiaohong

    2017-01-27

    A new series of diphenylpyrimidine derivatives (DPPYs) bearing various aniline side chains at the C-2 position of pyrimidine core were synthesized as potent BTK inhibitors. Most of these inhibitors displayed improved activity against B leukemia cell lines compared with lead compound spebrutinib. Subsequent studies showed that the peculiar inhibitor 7j, with IC50 values of 10.5 μM against Ramos cells and 19.1 μM against Raji cells, also displayed slightly higher inhibitory ability than the novel agent ibrutinib. Moreover, compound 7j is not sensitive to normal cells PBMC, indicating low cell cytotoxicity. In addition, flow cytometry analysis indicated that 7j significantly induced the apoptosis of Ramos cells, and arrested the cell cycle at the G0/G1 phase. These explorations provided new clues to discover pyrimidine scaffold as more effective BTK inhibitors.

  15. Apoptosis induced by the Tibetan herbal remedy PADMA 28 in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2

    PubMed Central

    Jenny, Marcel; Schwaiger, Wolfgang; Bernhard, David; Wrulich, Oliver A; Cosaceanu, Daria; Fuchs, Dietmar; Ueberall, Florian

    2005-01-01

    The Tibetan herbal remedy PADMA 28 revealed promising results to support treatment of atherosclerosis, Charot syndrome (intermittent claudication), chronic active hepatitis and infection of the respiratory tract. The remedy was confirmed to be closely linked with anti- and pro-oxidative properties in vitro. In this study, apoptogenic and survival effects of PADMA 28 were investigated in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2. PADMA 28 led to a concentration-dependent inhibition of cell proliferation accompanied by the accumulation of CEM-C7H2 cells in subG1 phase, fragmentation of poly (ADP-ribose) polymerase (PARP) and nuclear body formation. Treatment with PADMA 28 rescued to some extent cells over-expressing Bcl-2 from apoptosis. This finding suggests that the mechanism of action of PADMA 28 may be via interference with Bcl-2 triggered survival pathways. PMID:16138918

  16. Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1.

    PubMed

    Nukuzuma, Souichi; Nakamichi, Kazuo; Kameoka, Masanori; Sugiura, Shigeki; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu

    2010-12-01

    The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.

  17. Pigment epithelium-derived factor (PEDF) regulates metabolism and insulin secretion from a clonal rat pancreatic beta cell line BRIN-BD11 and mouse islets.

    PubMed

    Chen, Younan; Carlessi, Rodrigo; Walz, Nikita; Cruzat, Vinicius Fernandes; Keane, Kevin; John, Abraham N; Jiang, Fang-Xu; Carnagarin, Revathy; Dass, Crispin R; Newsholme, Philip

    2016-05-05

    Pigment epithelium-derived factor (PEDF) is a multifunctional glycoprotein, associated with lipid catabolism and insulin resistance. In the present study, PEDF increased chronic and acute insulin secretion in a clonal rat β-cell line BRIN-BD11, without alteration of glucose consumption. PEDF also stimulated insulin secretion from primary mouse islets. Seahorse flux analysis demonstrated that PEDF did not change mitochondrial respiration and glycolytic function. The cytosolic presence of the putative PEDF receptor - adipose triglyceride lipase (ATGL) - was identified, and ATGL associated stimulation of glycerol release was robustly enhanced by PEDF, while intracellular ATP levels increased. Addition of palmitate or ex vivo stimulation with inflammatory mediators induced β-cell dysfunction, effects not altered by the addition of PEDF. In conclusion, PEDF increased insulin secretion in BRIN-BD11 and islet cells, but had no impact on glucose metabolism. Thus elevated lipolysis and enhanced fatty acid availability may impact insulin secretion following PEDF receptor (ATGL) stimulation.

  18. Design, Synthesis, and Antitumor Activity of Novel 5-Pyridyl-1,3,4-oxadiazole Derivatives against the Breast Cancer Cell Line MCF-7.

    PubMed

    Khalil, Nadia Abdalla; Kamal, Aliaa Moh; Emam, Soha Hussein

    2015-01-01

    Various 1,3,4-oxadiazole-2-thiol derivatives have considerable potential in the field of antitumor activity. On the basis of the structure of the highly active reported oxadiazole analogues, 36 novel compounds were designed. Their molecular transport properties were predicted using a computer-aided program, and they were then synthesized and tested for anticancer activity against the breast cancer cell line MCF-7. Most of the tested compounds showed excellent to potent cytotoxic activity. Docking studies were carried out to examine the possibilities of the target compounds to become lead compounds in the future after more biological investigations. Compounds 18 and 22 were more active than the reference drug with IC₅₀ values of 0.010 µM and 0.012 µM, respectively, and binding energy scores of -10.32 and -10.25, respectively.

  19. Molecular association of 2-(n-alkylamino)-1,4-naphthoquinone derivatives: Electrochemical, DFT studies and antiproliferative activity against leukemia cell lines

    NASA Astrophysics Data System (ADS)

    Patil, Rishikesh; Bhand, Sujit; Konkimalla, V. Badireenath; Banerjee, Priyabrata; Ugale, Bharat; Chadar, Dattatray; Saha, Sourav Kr.; Praharaj, Prakash Priyadarshi; Nagaraja, C. M.; Chakrovarty, Debamitra; Salunke-Gawali, Sunita

    2016-12-01

    Molecular structures and their molecular association of 2-(n-alkylamino)-1,4-naphthoquinone, viz., LH-3; propyl, LH-4; butyl and LH-8; octyl derivatives were studied by single crystal X-ray diffraction studies. Synthesis and characterization of 2-octylamino-1,4-naphthoquinone; LH-8 was discussed. The molecule of LH-3 crystallizes in orthorhombic space group P21/c, while the LH-4 and LH-8 molecule crystallizes in triclinic space group P-1. LH-3, LH-4 and LH-8 showed intermolecular N-H⋯O and C-H⋯O interactions, LH-3 showed unique C(3)-H(3)⋯O(1) interaction. Interchain π-π stacking, slipped π-π stacking and C⋯O close contacts was respectively observed in LH-3, LH-4 and LH-8. Electrochemical studies were performed on first eight members of homologous series of 2-(n-alkylamino)-1,4-naphthoquinone (LH-1 to LH-8) by cyclic voltammetry. Naphthoquinone to naphthosemiquinone reversible redox couple was observed in all compounds ∼ E1/2 = -0.657 ± 0.05 V. HOMO-LUMO band gap was determined for the neutral form as well as the monoanionic radical form viz. naphthosemiquinone form of selected derivatives by DFT studies. It has been observed that the electron density is delocalized in the naphthoquinone ring in both neutral as well as one electron reduced form of compounds. Antiproliferative activity of LH-1 to LH-8 was evaluated against two cancer cell lines, THP1(acute monocytic leukemia) and K562(human immortalized myelogenous leukemia cell line) cells. It was observed that, in THP1 cells, compounds LH-2 and LH-3 are very active while LH-1, LH-4 and LH-6 were moderately active and LH-5, LH-7 and LH-8 were totally inactive. Contrastingly, in K562 cells all of the compounds were moderately active.

  20. Proline modulates the effect of bisphosphonate on calcium levels and adenosine triphosphate production in cell lines derived from bovine Echinococcus granulosus protoscoleces.

    PubMed

    Fuchs, A G; Echeverría, C I; Pérez Rojo, F G; Prieto González, E A; Roldán, E J A

    2014-12-01

    Bisphosphonates have been proposed as pharmacological agents against parasite and cancer cell growth. The effect of these compounds on helminthic cell viability and acellular compartment morphology, however, has not yet been studied. The effects of different types of bisphosphonates, namely etidronate (EHDP), pamidronate (APD), alendronate (ABP), ibandronate (IB) and olpadronate (OPD), and their interaction with amiloride, 1,25-dihydroxycholecalciferol (D3) and proline were evaluated on a cell line derived from bovine Echinococcus granulousus protoscoleces (EGPE) that forms cystic colonies in agarose. The EGPE cell line allowed testing the effect of bisphosphonates alone and in association with other compounds that could modulate calcium apposition/deposition, and were useful in measuring the impact of these compounds on cell growth, cystic colony formation and calcium storage. Decreased cell growth and cystic colony formation were found with EHDP, IB and OPD, and increased calcium storage with EHDP only. Calcium storage in EGPE cells appeared to be sensitive to the effect of amiloride, D3 and proline. Proline decreased calcium storage and increased colony formation. Changes in calcium storage may be associated with degenerative changes of the cysts, as shown in the in vitro colony model and linked to an adenosine triphosphate (ATP) decrease. In conclusion, bisphosphonates could be suitable tempering drugs to treat cestode infections.

  1. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

    SciTech Connect

    Beretta, L.; Boutterin, M.C.; Sobel, A.

    1988-01-01

    We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and (/sup 32/P) phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins.

  2. Synthesis of 17β-N-arylcarbamoylandrost-4-en-3-one derivatives and their anti-proliferative effect on human androgen-sensitive LNCaP cell line.

    PubMed

    Cortés-Benítez, Francisco; Cabeza, Marisa; Ramírez-Apan, María Teresa; Alvarez-Manrique, Berenice; Bratoeff, Eugene

    2016-10-04

    In this study, we report the synthesis and anti-proliferative effect of a set of eight androst-4-ene-3-one derivatives with different arylcarbamoyl groups at C-17. The novel compounds were prepared from commercially available 3β-hydroxy-5-pregnen-20-one and evaluated against the androgen-sensitive human prostate adenocarcinoma LNCaP cell line. The cancerous cells were exposed to 50 μM of each compound and the proliferating agent testosterone (T) or dihydrotestosterone (DHT). The most potent compounds from this assay were further tested against the androgen-insensitive PC3 cell line. We also demonstrate the activity of these compounds on rat peripheral blood mononuclear cells for comparison. Both 17β-N-[3,5-bis(trifluoromethyl)phenylcarbamoyl]androst-4-ene-3-one (6f) and 17β-N-(1,3-thiazol-2-ylcarbamoyl)androst-4-ene-3-one (6g) exhibited a higher growth inhibitory effect than commercially available drugs finasteride, flutamide and ketoconazole on LNCaP cells in the presence and absence of androgens. In addition, 6f and 6g demonstrated high potency on PC3 cells suggesting an androgen-independent anti-proliferative effect. Moreover, the novel compounds showed a small effect on rat mononuclear cells, an indication of low toxicity.

  3. Effect of the nature of the spacer on gene transfer efficacies of novel thiocholesterol derived gemini lipids in different cell lines: a structure-activity investigation.

    PubMed

    Bajaj, Avinash; Kondaiah, Paturu; Bhattacharya, Santanu

    2008-04-24

    A structure-activity investigation was undertaken to see the effect of the nature of the spacer on the gene transfection efficacies of thiocholesterol-derived cationic gemini lipids possessing disulfide linkage between the cationic headgroup and the thiocholesterol moiety. Three gemini cationic lipids possessing hydrophobic flexible (-(CH 2) 5-; 1), hydrophobic rigid (-C 6H 4-; 2), and hydrophilic flexible (-CH 2-CH 2-O-CH 2-CH 2-; 3) spacer segments were synthesized. In HeLa cells, lipid formulations 1 and 2 were found to be more effective as compared to lipid 3 formulation. In HT1080 cell line, the order of transfectability was 3 > 1 > 2. Transfection studies in HeLa and HT1080 cell line also showed 40-50% transfection efficacy in the presence of 10% serum conditions. These formulations were also able to transfect gene across difficult cells like HaCaT. Cytotoxic studies showed the nontoxic nature of these lipid-DNA complexes at different N/P ratios used for transfection studies.

  4. Multidrug resistance-associated protein 3 (Mrp3/Abcc3/Moat-D) is expressed in the SAE Squalus acanthias shark embryo-derived cell line.

    PubMed

    Kobayashi, Hiroshi; Parton, Angela; Czechanski, Anne; Durkin, Christopher; Kong, Chi-Chon; Barnes, David

    2007-01-01

    The multidrug resistance-associated protein 3 (MRP3/Mrp3) is a member of the ATP-binding cassette (ABC) protein family of membrane transporters and related proteins that act on a variety of xenobiotic and anionic molecules to transfer these substrates in an ATP-dependent manner. In recent years, useful comparative information regarding evolutionarily conserved structure and transport functions of these proteins has accrued through the use of primitive marine animals such as cartilaginous fish. Until recently, one missing tool in comparative studies with cartilaginous fish was cell culture. We have derived from the embryo of Squalus acanthias, the spiny dogfish shark, the S. acanthias embryo (SAE) mesenchymal stem cell line. This is the first continuously proliferating cell line from a cartilaginous fish. We identified expression of Mrp3 in this cell line, cloned the molecule, and examined molecular and cellular physiological aspects of the protein. Shark Mrp3 is characterized by three membrane-spanning domains and two nucleotide-binding domains. Multiple alignments with other species showed that the shark Mrp3 amino acid sequence was well conserved. The shark sequence was overall 64% identical to human MRP3, 72% identical to chicken Mrp3, and 71% identical to frog and stickleback Mrp3. Highest identity between shark and human amino acid sequence (82%) was seen in the carboxyl-terminal nucleotide-binding domain of the proteins. Cell culture experiments showed that mRNA for the protein was induced as much as 25-fold by peptide growth factors, fetal bovine serum, and lipid nutritional components, with the largest effect mediated by a combination of lipids including unsaturated and saturated fatty acids, cholesterol, and vitamin E.

  5. Gene and microRNA modulation upon trabectedin treatment in a human intrahepatic cholangiocarcinoma paired patient derived xenograft and cell line

    PubMed Central

    Peraldo Neia, Caterina; Cavalloni, Giuliana; Chiorino, Giovanna; Ostano, Paola; Aglietta, Massimo; Leone, Francesco

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is an aggressive and lethal malignancy with limited therapeutic options. Trabectedin has a high antitumor activity in preclinical models of biliary tract carcinoma (BTC), being a promising alternative treatment. Here, we studied the effect of trabectedin at transcriptomic level on an ICC patient derived xenograft (PDX) and on the derived cell line, MT-CHC01. Further, putative targets of trabectedin were explored in the in vitro model. In vitro, trabectedin inhibited genes involved in protein modification, neurogenesis, migration, and motility; it induced the expression of genes involved in keratinization, tissues development, and apoptotic processes. In the PDX model, trabectedin affected ECM-receptor interaction, focal adhesion, complement and coagulation cascades, Hedgehog, MAPK, EGFR signaling via PIP3 pathway, and apoptosis. Among down-regulated genes, we selected SYK and LGALS1; their silencing caused a significantly reduction of migration, but did not affect proliferation in in vitro models. In MT-CHC01 cells, 24 microRNAs were deregulated upon drug treatment, while only 5 microRNAs were perturbed by trabectedin in PDX. The target prediction analysis showed that SYK and LGALS1 are putative targets of up-regulated microRNAs. In conclusion, we described that trabectedin affected genes and microRNAs involved in tumor progression and metastatic processes, reflecting data previously obtained at macroscopically level; in particular, we identified SYK and LGALS1 as new putative targets of trabectedin. PMID:27902465

  6. The role of WNT/β-catenin signaling pathway in melanoma epithelial-to-mesenchymal-like switching: evidences from patients-derived cell lines

    PubMed Central

    Kovacs, Daniela; Migliano, Emilia; Muscardin, Luca; Silipo, Vitaliano; Catricalà, Caterina; Picardo, Mauro; Bellei, Barbara

    2016-01-01

    Deregulations or mutations of WNT/β-catenin signaling have been associated to both tumour formation and progression. However, contradictory results concerning the role of β-catenin in human melanoma address an open question on its oncogenic nature and prognostic value in this tumour. Changes in WNT signaling pathways have been linked to phenotype switching of melanoma cells between a highly proliferative/non-invasive and a slow proliferative/metastatic condition. We used a novel panel of cell lines isolated from melanoma specimens, at initial passages, to investigate phenotype differences related to the levels and activity of WNT/β-catenin signaling pathway. This in vitro cell system revealed a marked heterogeneity that comprises, in some cases, two distinct tumour-derived subpopulations of cells presenting a different activation level and cellular distribution of β-catenin. In cells derived from the same tumor, we demonstrated that the prevalence of LEF1 (high β-catenin expressing cells) or TCF4 (low β-catenin expressing cells) as β-catenin partner for DNA binding, is associated to the expression of two distinct profiles of WNT-responsive genes. Interestingly, melanoma cells expressing relative low level of β-catenin and an invasive markers signature were associated to the TNF-α-induced pro-inflammatory pathway and to the chemotherapy resistance, suggesting that the co-existence of melanoma subpopulations with distinct biological properties could influence the impact of chemo- and immunotherapy. PMID:27175588

  7. Exposure to Nickel, Chromium, or Cadmium Causes Distinct Changes in the Gene Expression Patterns of a Rat Liver Derived Cell Line

    PubMed Central

    Permenter, Matthew G.; Lewis, John A.; Jackson, David A.

    2011-01-01

    Many heavy metals, including nickel (Ni), cadmium (Cd), and chromium (Cr) are toxic industrial chemicals with an exposure risk in both occupational and environmental settings that may cause harmful outcomes. While these substances are known to produce adverse health effects leading to disease or health problems, the detailed mechanisms remain unclear. To elucidate the processes involved in the toxicity of nickel, cadmium, and chromium at the molecular level and to perform a comparative analysis, H4-II-E-C3 rat liver-derived cell lines were treated with soluble salts of each metal using concentrations derived from viability assays, and gene expression patterns were determined with DNA microarrays. We identified both common and unique biological responses to exposure to the three metals. Nickel, cadmium, chromium all induced oxidative stress with both similar and unique genes and pathways responding to this stress. Although all three metals are known to be genotoxic, evidence for DNA damage in our study only exists in response to chromium. Nickel induced a hypoxic response as well as inducing genes involved in chromatin structure, perhaps by replacing iron in key proteins. Cadmium distinctly perturbed genes related to endoplasmic reticulum stress and invoked the unfolded protein response leading to apoptosis. With these studies, we have completed the first gene expression comparative analysis of nickel, cadmium, and chromium in H4-II-E-C3 cells. PMID:22110744

  8. NADPH oxidase-derived reactive oxygen species are essential for differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts.

    PubMed

    Sasaki, Hideyuki; Yamamoto, Hironori; Tominaga, Kumiko; Masuda, Kiyoshi; Kawai, Tomoko; Teshima-Kondo, Shigetada; Rokutan, Kazuhito

    2009-02-01

    Reactive oxygen species (ROS) derived from NADPH oxidase (Nox) homologues have been suggested to regulate osteoclast differentiation. However, no bone abnormalities have been documented in Nox1 deficient, Nox2 deficient, or Nox3 mutant mice. During receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts, mRNA levels of Nox enzymes (Nox1-4) and their adaptor proteins were monitored by real-time reverse transcriptase PCR. RAW264.7 cells constitutively expressed abundant Nox2 mRNA and small amounts of Nox1 and Nox3 transcripts. RANKL markedly attenuated Nox2 mRNA expression in association with reciprocal up-regulation of Nox1 and Nox3 transcripts. Introduction of small interference RNA targeting p67(phox) or p22(phox) into RAW264.7 cells effectively down-regulated ROS generation and significantly suppressed the RANKL-stimulated differentiation, which was assessed by appearance of tartrate resistant acid phosphatase (TRAP)-positive, multinucleated cells having an ability to form resorption pits on calcium phosphate thin film-coated disks, and by expression of osteoclast marker genes (TRAP, cathepsin K, Atp6i, ClC-7, and NFATc1). Our results suggest that RANKL may stimulate switching between Nox homologues during osteoclast differentiation, and Nox-derived ROS may be crucial for RANKL-induced osteoclast differentiation.

  9. Repair of chromosome damage induced by X-irradiation during G/sub 2/ phase in a line of normal human fibroblasts and its malignant derivative

    SciTech Connect

    Parshad, R.; Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

    1982-08-01

    A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G/sub 2/ phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or ..beta..-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G/sub 2/ phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, which destroys H/sub 2/O/sub 2/, or mannitol, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G/sub 2/ phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives.

  10. Organ heterogeneity of host-derived matrix metalloproteinase expression and its involvement in multiple-organ metastasis by lung cancer cell lines.

    PubMed

    Shiraga, Minoru; Yano, Seiji; Yamamoto, Akihiko; Ogawa, Hirohisa; Goto, Hisatsugu; Miki, Toyokazu; Miki, Keisuke; Zhang, Helong; Sone, Saburo

    2002-10-15

    Cancer metastasis is tightly regulated by the interaction of tumor cells and host organ microenvironments. Matrix metalloproteinases (MMPs), produced by both tumor cells and host stromal cells, play a central role in tumor invasion and angiogenesis. We determined whether metastatic potential of lung cancer to multiple organs is dependent solely on the expression of MMPs by tumor cells, using two metastasis models of human lung cancer cell lines expressing various levels of MMPs and a MMP inhibitor (ONO-4817). In the lung metastasis model, tumor cells (PC14, PC14PE6, H226, A549) inoculated i.v. into nude or SCID mice metastasized only in the lung. In the multiple-organ metastasis model, tumor cells (RERF-LC-AI, SBC-3/DOX, H69/VP, which express low levels of MMPs) inoculated i.v. into natural killer cell-depleted SCID mice metastasized into the liver, kidneys, and systemic lymph nodes. Film in situ zymography analysis revealed that the nontumor parenchyma of the lung had no gelatinolytic activity, whereas gelatinolytic activity of the liver and kidney was high and low, respectively. In the lung metastasis model, gelatinolytic activity of lung nodules directly correlated with the in vitro expression of MMP-2 and MMP-9 by tumor cells. Inhibition of MMP activity by ONO-4817 suppressed lung metastasis by the cell lines that expressed MMPs, but not those that did not express MMP, via the inhibition of MMP activity of lung tumors. In the multiple-organ metastasis model, liver parenchyma, but not liver nodules, showed gelatinolytic activity. The MMP inhibition reduced metastasis to the liver, but not to the kidney or lymph nodes, via inhibition of MMP activity of liver parenchyma. These findings suggest that MMP expression varies among the host organ microenvironments and that stromal MMPs may promote metastasis of lung cancer. Therefore, antimetastatic effects based on MMP inhibition may be dependent on MMPs derived not only from tumor cells but also from organ

  11. Capability of human umbilical cord blood progenitor-derived endothelial cells to form an efficient lining on a polyester vascular graft in vitro.

    PubMed

    Bérard, Xavier; Rémy-Zolghadri, Murielle; Bourget, Chantal; Turner, Neill; Bareille, Reine; Daculsi, Richard; Bordenave, Laurence

    2009-05-01

    One of the goals of vascular tissue engineering is to create functional conduits for small-diameter bypass grafting. The present biocompatibility study was undertaken to check the ability of cord blood progenitor-derived endothelial cells (PDECs) to take the place of endothelial cells in vascular tissue engineering. After isolation, culture and characterization of endothelial progenitor cells, the following parameters were explored, with a commercial knitted polyester prosthesis (Polymaille C, Laboratoires Pérouse, France) impregnated with collagen: cell adhesion and proliferation, colonization, cell retention on exposure to flow, and the ability of PDECs to be regulated by arterial shear stress via mRNA levels. PDECs were able to adhere to commercial collagen-coated vascular grafts in serum-free conditions, and were maintained but did not proliferate when seeded at 2.0 x 10(5) cm(-2). Cellularized conduits were analyzed by histology and histochemical staining, demonstrating collagen impregnation and the endothelial characteristics of the colonizing cells. Thirty-six hours after cell seeding the grafts were maintained for 6 h of either static conditions (controls) or application of pulsatile laminar shear stress, which restored the integrity of the monolayer. Finally, quantitative real-time RT-PCR analysis performed at 4 and 8 h from cells lining grafts showed that MMP1 mRNA only was increased at 4h whereas vWF, VE-cadherin and KDR were not significantly modified at 4 and 8 h. Our results show that human cord blood PDECs are capable of forming an efficient lining and to withstand shear stress.

  12. Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout–derived cell line, RTG-2

    USGS Publications Warehouse

    Kurath, Gael; Purcell, Maureen K.; Wargo, Andrew; Park, Jeong Woo; Moon, Chang Hoon

    2010-01-01

    Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout–derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

  13. Inhibition of Mevalonate Pathway and Synthesis of the Storage Lipids in Human Liver-Derived and Non-liver Cell Lines by Lippia alba Essential Oils.

    PubMed

    Montero-Villegas, Sandra; Polo, Mónica; Galle, Marianela; Rodenak-Kladniew, Boris; Castro, María; Ves-Losada, Ana; Crespo, Rosana; García de Bravo, Margarita

    2017-01-01

    The essential oils (EOs) of Lippia alba, an herb extensively used as a folk medicine in Latin America, are today promoted as an effective means of eliminating problems caused by hyperlipemia. We hypothesized that L.alba EOs inhibited cholesterol and triacylglycerols synthesis and decreased the intracellular depots of those lipids (lipid droplets), mechanisms involving the induction of a hypolipidemic response. Our aim was, therefore, to evaluate the hypolipogenic capability of the EOs of four L. alba chemotypes on liver-derived (HepG2) and non-liver (A549) human cell lines and to identify the potential biochemical targets of those chemotypes, particularly within the mevalonate pathway (MP). [(14)C]Acetate was used as radioactive precursor for assays. Lipid analyses were performed by thin-layer and capillary gas chromatography, lipid droplets analyzed by fluorescence microscopy, and HMGCR levels determined by Western blot. In both cell lines, all four chemotypes exerted hypocholesterogenic effects within a concentration range of 3.2-32 µg/mL. Nonsaponifiable lipids manifested a decrease in incorporation of [(14)C]acetate into squalene, lanosterol, lathosterol, and cholesterol, but not into ubiquinone, thus suggesting an inhibition of enzymes in the MP downstream from farnesyl pyrophosphate. The tagetenone chemotype, the most efficacious hypocholesterogenic L. alba EO, lowered HMGCR protein levels; inhibited triacylglycerols, cholesteryl esters, and phospholipids synthesis; and diminished lipid droplets in size and volume. These results revealed that L. alba EOs inhibited different lipogenic pathways and such lipid-lowering effects could prove essential to prevent cardiovascular diseases.

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

    PubMed

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

    2007-08-01

    Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor-like role in developed human retina is unknown. 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. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor-2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including betaIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S-opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell-based therapies to restore retinal function. Disclosure of potential conflicts of interest is found at the end of this article.

  15. Differential growth of U and M type infectious haematopoietic necrosis virus in a rainbow trout-derived cell line, RTG-2.

    PubMed

    Park, J W; Moon, C H; Wargo, A R; Purcell, M K; Kurath, G

    2010-07-01

    Infectious haematopoietic necrosis virus (IHNV) is one of the most important viral pathogens of salmonids. In rainbow trout, IHNV isolates in the M genogroup are highly pathogenic, while U genogroup isolates are significantly less pathogenic. We show here that, at a multiplicity of infection (MOI) of 1, a representative U type strain yielded 42-fold less infectious virus than an M type strain in the rainbow trout-derived RTG-2 cell line at 24 h post-infection (p.i.). However, at an MOI of 10, there was only fivefold difference in the yield of infectious virus between the U and M strains. Quantification of extracellular viral genomic RNA suggested that the number of virus particles released from cells infected with the U strain at a MOI of 1 was 47-fold lower than from M-infected cells, but U and M virions were equally infectious by particle to infectivity ratios. At an MOI of 1, U strain intracellular viral genome accumulation and transcription were 37- and 12-fold lower, respectively, than those of the M strain at 24 h p.i. Viral nucleocapsid (N) protein accumulation in U strain infections was fivefold lower than in M strain infections. These results suggest that the block in U type strain growth in RTG-2 cells was because of the effects of reduced genome replication and transcription. The reduced growth of the U strain does not seem to be caused by defective genes, because the U and M strains grew equally well in the permissive epithelioma papulosum cyprini cell line at an MOI of 1. This suggests that host-specific factors in RTG-2 cells control the growth of the IHNV U and M strains differently, leading to growth restriction of the U type virus during the RNA synthesis step.

  16. Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.

    PubMed

    Tsui, Cynthia C; Gabreski, Nicole A; Hein, Sarah J; Pierchala, Brian A

    2015-09-23

    Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. Significance statement: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex

  17. Apoptotic induction by pinobanksin and some of its ester derivatives from Sonoran propolis in a B-cell lymphoma cell line.

    PubMed

    Alday, Efrain; Valencia, Dora; Carreño, Ana Laura; Picerno, Patrizia; Piccinelli, Anna Lisa; Rastrelli, Luca; Robles-Zepeda, Ramon; Hernandez, Javier; Velazquez, Carlos

    2015-12-05

    Propolis is a resinous substance produced by honeybees (Apis mellifera) from the selective collection of exudates and bud secretions from several plants. In previous works, we reported the antiproliferative activity of Sonoran propolis (SP) on cancer cells; in addition we suggested the induction of apoptosis after treatment with SP due to the presence of morphological changes and a characteristic DNA fragmentation pattern. Herein, in this study we demonstrated that the antiproliferative effect of SP is induced through apoptosis in a B-cell lymphoma cancer cell line, M12.C3.F6, by an annexin V-FITC/Propidium iodide double labeling. This apoptotic effect of SP resulted to be mediated by modulations in the loss of mitochondrial membrane potential (ΔΨm) and through activation of caspases signaling pathway (3, 8 and 9). Afterward, in order to characterize the chemical constituents of SP that induce apoptosis in cancer cells, an HPLC-PDA-ESI-MS/MS method followed by a preparative isolation procedure and NMR spectroscopy analysis have been used. Eighteen flavonoids, commonly described in propolis from temperate regions, were characterized. Chrysin, pinocembrin, pinobanksin and its ester derivatives are the main constituents of SP and some of them have never been reported in SP. In addition, two esters of pinobanksin (8 and 13) are described by first time in propolis samples in general. The antiproliferative activity on M12.C3.F6 cells through apoptosis induction was exhibited by pinobanksin (4), pinobanksin-3-O-propanoate (14), pinobanksin-3-O-butyrate (16), pinobanksin-3-O-pentanoate (17), and the already reported galangin (11), chrysin (9) and CAPE. To our knowledge this is the first report of bioactivity of pinobanksin and some of its ester derivatives as apoptosis inducers. Further studies are needed to advance in the understanding of the molecular basis of apoptosis induction by SP and its constituents, as well as the structure-activity relationship of them.

  18. Establishment and cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker.

    PubMed

    Wu, Shuai Shuai; Li, Dong Jiang; Lv, Chun Rong; Yang, Hong Yuan; Zhu, Lan; Li, Wei Juan; Quan, Guo Bo; Hong, Qiong Hua

    2013-01-01

    In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1 : 1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n = 54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50 mM, the viable percentage of frozen-thawed fibroblast cells was 91.93% +/- 2.24% and 94.13% +/- 0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10% +/- 1.49%, P < 0.05). In conclusion, the skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in

  19. Cytotoxicity and cell death mechanisms induced by a novel bisnaphthalimidopropyl derivative against the NCI-H460 non-small lung cancer cell line.

    PubMed

    Lima, Raquel T; Barron, Gemma A; Grabowska, Joanna A; Bermano, Giovanna; Kaur, Simranjeet; Roy, Nilanjan; Vasconcelos, M Helena; Lin, Paul K T

    2013-03-01

    Some polyamine derivatives, namely the bisnaphthalimidopropyl polyamines (BNIPPs) may have potential as anticancer drugs. Indeed, previous work from some of us had shown that the ability of these molecules to bind to DNA may contribute to their cytotoxicity. However, their precise mode of action has not been fully understood. In the present work, we report for the first time the effect of the previously synthesised compounds, BNIPDaCHM and NPA, together with a new BNIP derivative (BNIP-3,4-DaDPM) in the in vitro growth of a non-small cell lung cancer cell line (NCI-H460). In addition, for the most potent compound (BNIPDaCHM), its activity as sirtuin inhibitor was investigated in vitro and further confirmed in silico. Results in the NCI-H460 cells showed that, from the compounds tested, BNIPDaCHM was the most potent (GI50 of 1.3 μM). In addition, a concentration-dependent alteration in the normal NCI-H460 cell cycle profile was observed following treatment with BNIPDaCHM as well as an increase in the sub-G1 peak (suggestive of apoptotis). This effect was further supported by Annexin V/PI staining and by analysing the expression of proteins related to apoptosis (cleaved PARP and Caspase-3) by Western blot. It was also observed that BNIPDaCHM inhibited the activity of SIRT2 in vitro, but not of SIRT1. Accordingly, this compound also caused a small increase in tubulin acetylation in NCI-H460 cells. To determine the binding potential of BNIPDaCHM on hSIRT2 and to further validate its inhibitory action, in silico docking studies were carried out, which revealed that BNIPDaCHM is composed of an entirely new SIRT2- inhibiting structural scaffold. In conclusion, this study indicates that BNIP derivatives with a novel structural backbone, such as BNIPDaCHM, may have potential as building blocks for novel antitumour agents which might selectively bind to hSIRT-2.

  20. Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-γ expression

    PubMed Central

    Mwangi, Simon Musyoka; Peng, Sophia; Nezami, Behtash Ghazi; Thorn, Natalie; Farris, Alton B.; Jain, Sanjay; Laroui, Hamed; Merlin, Didier; Anania, Frank

    2015-01-01

    Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease. PMID:26564715

  1. EB66 cell line, a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity

    PubMed Central

    Olivier, Stéphane; Jacoby, Marine; Brillon, Cédric; Bouletreau, Sylvana; Mollet, Thomas; Nerriere, Olivier; Angel, Audrey; Danet, Sévérine; Souttou, Boussad; Guehenneux, Fabienne; Gauthier, Laurent; Berthomé, Mathilde; Vié, Henri; Beltraminelli, Nicola

    2010-01-01

    Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of monoclonal antibodies and to potentially reduce the therapeutic cost. We report here that the EB66 cell line derived from duck embryonic stem cells can be efficiently genetically engineered to produce mAbs at yields beyond a 1 g/L, as suspension cells grown in serum-free culture media. EB66 cells display additional attractive growth characteristics such as a very short population doubling time of 12–14 h, a capacity to reach very high cell density (>30 million cells/mL) and a unique metabolic profile resulting in low ammonium and lactate accumulation and low glutamine consumption, even at high cell densities. Furthermore, mAbs produced on EB66 cells display a naturally reduced fucose content resulting in strongly enhanced ADCC activity. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies. PMID:20562528

  2. Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2.

    PubMed

    Liu, Yong; Liu, Wen-bin; Liu, Kai-jun; Ao, Lin; Zhong, Julia Li; Cao, Jia; Liu, Jin-yi

    2015-01-01

    Previous studies have shown that the male reproductive system is one of the most sensitive organs to electromagnetic radiation. However, the biological effects and molecular mechanism are largely unclear. Our study was designed to elucidate the epigenetic effects of 50 Hz ELF-EMF in vitro. Mouse spermatocyte-derived GC-2 cell line was exposed to 50 Hz ELF-EMF (5 min on and 10 min off) at magnetic field intensity of 1 mT, 2 mT, and 3 mT with an intermittent exposure for 72 h. We found that 50 Hz ELF-EMF exposure decreased genome-wide methylation at 1 mT, but global methylation was higher at 3 mT compared with the controls. The expression of DNMT1 and DNMT3b was decreased at 1 mT, and 50 Hz ELF-EMF can increase the expression of DNMT1 and DNMT3b of GC-2 cells at 3 mT. However, 50 Hz ELF-EMF had little influence on the expression of DNMT3a. Then, we established DNA methylation and gene expression profiling and validated some genes with aberrant DNA methylation and expression at different intensity of 50 Hz ELF-EMF. These results suggest that the alterations of genome-wide methylation and DNMTs expression may play an important role in the biological effects of 50 Hz ELF-EMF exposure.

  3. Inversely repeating integrated hepatitis B virus DNA and cellular flanking sequences in the human hepatoma-derived cell line huSP.

    PubMed Central

    Mizusawa, H; Taira, M; Yaginuma, K; Kobayashi, M; Yoshida, E; Koike, K

    1985-01-01

    Among recombinant phages carrying integrated hepatitis B virus (HBV) DNA sequences cloned from the human hepatoma-derived cell line huSP, one clone, lambda hu-489, revealed some unusual features. The 2.25-kilobase Eco D fragment from the insert of this clone hybridized to the HBV DNA probe only and its nucleotide sequence was determined. The viral sequence, as well as a cellular flanking sequence, showed extensive rearrangement accompanied by inverted repetition. The Eco D fragment contained HBV DNA from the 5'-end region of gene S to the middle of gene X, followed by a long cellular flanking sequence. Moreover, a part of gene X was found inversely repeated at the head of the same gene S in a head-to-head configuration truncated by the same cellular sequence. Therefore, the same junction sequence of viral DNA and the cellular sequence was found at two different sites in the Eco D fragment in opposite polarities. Images PMID:2982143

  4. Harpagoside attenuates MPTP/MPP⁺ induced dopaminergic neurodegeneration and movement disorder via elevating glial cell line-derived neurotrophic factor.

    PubMed

    Sun, Xiaoyu; Xiong, Zhongkui; Zhang, Yongfang; Meng, Ya; Xu, Gang; Xia, Zhiming; Li, Jiamei; Zhang, Rui; Ke, Zunji; Xia, Zongqin; Hu, Yaer

    2012-03-01

    Parkinson's disease is a chronic neurodegenerative movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. New therapeutic approaches aiming at delaying or reversing the neurodegenerative process are under active investigations. In this work, we found that harpagoside, an iridoid purified from the Chinese medicinal herb Scrophularia ningpoensis, could not only prevent but also rescue the dopaminergic neurodegeneration in MPTP/MPP(+) intoxication with promising efficacy. Firstly, in cultured mesencephalic neurons, harpagoside significantly attenuated the loss of TH-positive neuron numbers and the shortening of axonal length. Secondly, in a chronic MPTP mouse model, harpagoside dose-dependently improved the loco-motor ability (rotarod test), increased the TH-positive neuron numbers in the substantia nigra pars compacta (unbiased stereological counting) and increased the striatal DAT density ((125) I-FP-CIT autoradiography). Thirdly, harpagoside markedly elevated the GDNF mRNA and GDNF protein levels in MPTP/MPP(+) lesioned models. However, the protecting effect of harpagoside on the dopaminergic degeneration disappeared when the intrinsic GDNF action was blocked by either the Ret inhibitor PP1 or the neutralizing anti-GDNF antibody. Taken together, we conclude that harpagoside attenuates the dopaminergic neurodegeneration and movement disorder mainly through elevating glial cell line-derived neurotrophic factor.

  5. Activated microglia provide a neuroprotective role by balancing glial cell-line derived neurotrophic factor and tumor necrosis factor-α secretion after subacute cerebral ischemia.

    PubMed

    Wang, Jianping; Yang, Zhitang; Liu, Cong; Zhao, Yuanzheng; Chen, Yibing

    2013-01-01

    Microglia are the major immune cells in the central nervous system and play a key role in brain injury pathology. However, the role of activated microglia after subacute cerebral ischemia (SCI) remains unknown. To address this issue, we established a permanent middle cerebral artery occlusion (pMCAO) rat model and treated pMCAO rats with N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) (an inhibitor of microglial activation), or with vehicle alone. Finally, we determined the differences between the PJ34-and vehicle-treated rats with respect to neurological deficits, infarct volume, neuronal loss and the expression of CD11b (a marker of microglial activation), glial cell line-derived neurotrophic factor (GDNF) and tumor necrosis factor-α (TNF-α) at 1, 3 and 7 days after treatment. We found that the PJ34-treated rats had more severe neurological deficits and a larger infarct volume and exhibited a decreased CD11b expression, more neuronal loss, decreased expression of GDNF mRNA and protein but increased expression of TNF-α mRNA and protein compared with the vehicle-treated rats at 3 and 7 days after treatment. These results indicate that activated microglia provide a neuroprotective role through balancing GDNF and TNF-α expression following SCI.

  6. SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR

    PubMed Central

    Stabley, Deborah L; Harris, Ashlee W; Holbrook, Jennifer; Chubbs, Nicholas J; Lozo, Kevin W; Crawford, Thomas O; Swoboda, Kathryn J; Funanage, Vicky L; Wang, Wenlan; Mackenzie, William; Scavina, Mena; Sol-Church, Katia; Butchbach, Matthew E R

    2015-01-01

    Proximal spinal muscular atrophy (SMA) is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient-derived cell lines, the assay confirmed a strong inverse correlation between SMN2 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples. PMID:26247043

  7. Viral delivery of glial cell line-derived neurotrophic factor improves behavior and protects striatal neurons in a mouse model of Huntington’s disease

    PubMed Central

    McBride, Jodi L.; Ramaswamy, Shilpa; Gasmi, Mehdi; Bartus, Raymond T.; Herzog, Christopher D.; Brandon, Eugene P.; Zhou, Lili; Pitzer, Mark R.; Berry-Kravis, Elizabeth M.; Kordower, Jeffrey H.

    2006-01-01

    Huntington’s disease (HD) is a fatal, genetic, neurological disorder resulting from a trinucleotide repeat expansion in the gene that encodes for the protein huntingtin. These excessive repeats confer a toxic gain of function on huntingtin, which leads to the degeneration of striatal and cortical neurons and a devastating motor, cognitive, and psychological disorder. Trophic factor administration has emerged as a compelling potential therapy for a variety of neurodegenerative disorders, including HD. We previously demonstrated that viral delivery of glial cell line-derived neurotrophic factor (GDNF) provides structural and functional neuroprotection in a rat neurotoxin model of HD. In this report we demonstrate that viral delivery of GDNF into the striatum of presymptomatic mice ameliorates behavioral deficits on the accelerating rotorod and hind limb clasping tests in transgenic HD mice. Behavioral neuroprotection was associated with anatomical preservation of the number and size of striatal neurons from cell death and cell atrophy. Additionally, GDNF-treated mice had a lower percentage of neurons containing mutant huntingtin-stained inclusion bodies, a hallmark of HD pathology. These data further support the concept that viral vector delivery of GDNF may be a viable treatment for patients suffering from HD. PMID:16751280

  8. Study of orexins signal transduction pathways in rat olfactory mucosa and in olfactory sensory neurons-derived cell line Odora: multiple orexin signalling pathways.

    PubMed

    Gorojankina, Tatiana; Grébert, Denise; Salesse, Roland; Tanfin, Zahra; Caillol, Monique

    2007-06-07

    Orexins A and B (OxA and OxB) are multifunctional neuropeptides implicated in the regulation of energy metabolism, wakefulness but also in a broad range of motivated behaviours. They signal through two G-protein-coupled receptors: orexin receptor 1 and 2 (Ox1R and Ox2R). The orexins and their receptors are present at all levels of the rat olfactory system: epithelium, bulb, piriform cortex but their signalling mechanisms remain unknown. We have studied orexins signal transduction pathways in the rat olfactory mucosa (OM) and in the Odora cell line derived from olfactory sensory neurons and heterologously expressing Ox1R or Ox2R. We have demonstrated by western blot and RT-PCR that multiple components of adenylyl cyclase (AC) and phospholipase C (PLC) signalling pathways were identical in OM and Odora cells. OxA and OxB induced a weak increase in IP3 in OM; they induced a significant rise in cAMP and IP3 in Odora transfected cells, suggesting the activation of AC and PLC pathways. Both OxA and OxB induced intracellular calcium elevation and transient activation of MAP kinases (ERK42/44) in Odora/Ox1R and Odora/Ox2R cells. These results suggest the existence of multiple orexins signalling pathways in Odora cells and probably in OM, corresponding to different possible roles of these peptides.

  9. Age-related alterations in the expression of glial cell line-derived neurotrophic factor in the senescence-accelerated mouse brain.

    PubMed

    Miyazaki, Hiroyuki; Okuma, Yasunobu; Nomura, Jun; Nagashima, Kazuo; Nomura, Yasuyuki

    2003-05-01

    Senescence-accelerated mouse prone 8 (SAMP8) and prone 10 (SAMP10) are useful murine model of accelerated aging. SAMP8 shows marked impairment of learning and memory, whereas SAMP10 shows brain atrophy and aging-associated depressive behavior. This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) in SAMP8 and SAMP10 brains, relative to that in SAM resistant 1 (SAMR1) controls, which age normally. Hippocampal GDNF mRNA expression decreased in an age-dependent manner (10- vs 2-month-old animals) in the SAMR1, but not in the SAMP8 or SAMP10 strains. Furthermore, GDNF mRNA expression in 2-month-old SAMP8 and SAMP10 strains was less than in SAMR1 specimens of the same age. The number of surviving neurons in the CA1 region decreased with age in SAMP8 and SAMP10, and also decreased relative to the number of neurons in 10-month-old SAMR1 controls. Immunohistochemistry revealed that cells that were positive for GDNF-like activity in 10-month-old SAMP8 and SAMP10 were diffusely distributed, in part, around the pyramidal cell layer in the hippocampus. These findings suggest that low GDNF expression in young SAMP8 and SAMP10 may be involved in hippocampal dysfunctions, such as age-related learning impairment and neuronal death.

  10. Glial cell-line derived neurotrophic factor (GDNF) concentrations in cerebrospinal fluid and serum of patients with early Alzheimer's disease and normal controls.

    PubMed

    Straten, Guido; Eschweiler, Gerhard W; Maetzler, Walter; Laske, Christoph; Leyhe, Thomas

    2009-01-01

    As neurotrophic factors play an important role in development and maintenance of global central nervous system (CNS) function, we supposed that glial cell-line derived neurotrophic factor (GDNF), which has been extensively studied for its survival promoting effects especially concerning catecholaminergic neurons, also plays a significant role in neurodegenerative disease characterized mainly by damage of cholinergic CNS neurons like AD. Here we compared GDNF concentrations in serum and cerebrospinal fluid (CSF) of patients with probable Alzheimer's disease (AD) and normal controls (NC). While GDNF concentrations in CSF were significantly increased in patients with AD (291.7 +/- 85.8 pg/ml) compared with NC subjects (218.7 +/- 93.3 pg/ml, p = 0.012), GDNF concentration of AD patients (486.5 +/- 72.3 pg/ml) in serum were significantly decreased compared with the NC group (711.5 +/- 186.5 pg/ml, p < 0.001). Increased GDNF in CSF of AD might be due to an upregulated expression in CNS as an adaptive process of the impaired brain to enhance neurotrophic support at least in early stages of disease and/or impairment of CSF turnover. Decreased serum concentration of GDNF might be related to altered function of the blood brain barrier thus disturbing clearance or facilitating passover of potentially harmful metabolites.

  11. Technical advance: Langerhans cells derived from a human cell line in a full-thickness skin equivalent undergo allergen-induced maturation and migration.

    PubMed

    Ouwehand, Krista; Spiekstra, Sander W; Waaijman, Taco; Scheper, Rik J; de Gruijl, Tanja D; Gibbs, Susan

    2011-11-01

    In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

  12. The inducibility of human cytochrome P450 1A by environmental-relevant xenobiotics in the human hepatoma derived cell line HepG2.

    PubMed

    Rudzok, Susanne; Schmücking, Eike; Graebsch, Carolin; Herbarth, Olf; Bauer, Mario

    2009-11-01

    Overexpression of the CYP1 family, independent of gender, is focal to the evaluation of the risk of human cancer. We have analysed the ability of 17 anthropogenic environmental xenobiotics widely used in Europe within households and agriculture to induce the human cytochrome P450 1A (CYP1A) in the human hepatoma derived cell line HepG2. The xenobiotics were potent to concomitantly induce both CYP1A mRNA and CYP1A activity in a dose-response relationship. Exceptions were shown by the organophosphate insecticide chlorpyrifos and the imidazole fungicide prochloraz in high concentrations which were capable of both inhibiting the basal or abolishing the initially induced CYP1A activity, respectively. A CYP1A induction has been shown for the first time by the aromatic xenobiotics irgasan, permethrin and azoxystrobin, the nonaromatic tributyltinoxide and for humans by the piperonylbutoxide. The xenobiotics additionally differed by their induced CYP1A isoenzyme pattern. A pronounced CYP1A1 and CYP1A2 mRNA induction was given by the phenyl urea herbicide diuron and benzodiazole insecticide piperonylbutoxide, respectively. In conclusion, out of the environmental xenobiotics, we described new members of human CYP1A inducers which extend chemical structures of biotransformation activators.

  13. Glial cell line-derived neurotrophic factor is a survival factor for isolectin B4-positive, but not vanilloid receptor 1-positive, neurons in the mouse.

    PubMed

    Zwick, Melissa; Davis, Brian M; Woodbury, C Jeffrey; Burkett, John N; Koerber, H Richard; Simpson, James F; Albers, Kathryn M

    2002-05-15

    Most, if not all, nociceptor sensory neurons are dependent on nerve growth factor (NGF) during early embryonic development. A large subpopulation of these sensory neurons loses NGF dependency between embryonic day 16 and postnatal day 14 and become responsive to glial cell line-derived growth factor (GDNF), a member of the transforming growth factor beta (TGF-beta) family. To examine the survival and phenotypic effects of GDNF on sensory neurons in vivo, we generated transgenic mice that overexpress GDNF in the skin. GDNF-overexpresser mice had increased numbers of small unmyelinated sensory neurons that express the tyrosine kinase receptor Ret and bind the plant isolectin B4 (IB4). Surprisingly, in wild-type and transgenic mice, few ( approximately 2%) IB4-positive neurons expressed the vanilloid receptor VR1, a heat-sensitive receptor expressed by many IB4-positive neurons of the rat. Thus, in mouse, GDNF-dependent IB4-positive neurons must use a non-VR1 heat receptor. In addition, the behavior of GDNF-overexpresser animals to noxious heat or mechanical stimuli was indistinguishable from wild-type animals, indicating that, on a behavioral level, peripherally applied GDNF does not alter the sensitivity of the somatosensory system.

  14. Glial cell line-derived neurotrophic factor family ligands enhance capsaicin-stimulated release of calcitonin gene-related peptide from sensory neurons.

    PubMed

    Schmutzler, B S; Roy, S; Hingtgen, C M

    2009-06-16

    The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are a group of peptides that have been implicated as important factors in inflammation, since they are released in increased amounts during inflammation and induce thermal hyperalgesia upon injection. Mouse isolated sensory neurons in culture and freshly dissociated spinal cord slices were used to examine the enhancement in stimulated-release of the neuropeptide, calcitonin gene-related peptide (CGRP), as a measure of sensitization. Exposure of isolated sensory neurons in culture to GDNF, neurturin, and artemin enhanced the capsaicin-stimulated release of immunoreactive calcitonin gene-related peptide (iCGRP) two- to threefold, but did not increase potassium-stimulated release of iCGRP. A similar profile of sensitization was observed in freshly dissociated spinal cord slices. Persephin, another member of the GFL family thought to be important in development, was unable to induce an enhancement in the release of iCGRP. These results demonstrate that specific GFLs are important mediators affecting sensory neuronal sensitivity, likely through modulation of the capsaicin receptor. The sensitization of sensory neurons during inflammation, and the pain and neurogenic inflammation resulting from this sensitization, may be due in part to the effects of these selected GFLs.

  15. Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors

    PubMed Central

    Jaalouk, Diana E; Crosato, Milena; Brodt, Pnina; Galipeau, Jacques

    2006-01-01

    Background Self-inactivating retroviral vectors (SIN) are often associated with very low titers. Promoter elements embedded within SIN designs may suppress transcription of packageable retroviral RNA which in turn results in titer reduction. We tested whether this dominant-negative effect involves histone acetylation state. We designed an MLV-derived SIN vector using the cytomegalovirus immediate early enhancer-promoter (CMVIE) as an embedded internal promoter (SINCMV) and transfected the pantropic 293GPG packaging cell line. Results The SINCMV retroviral producer had uniformly very low titers (~10,000 infectious retroparticles per ml). Northern blot showed low levels of expression of retroviral mRNA in producer cells in particular that of packageable RNA transcript. Treatment of the producers with the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A reversed transcriptional suppression and resulted in an average 106.3 ± 4.6 – fold (P = 0.002) and 15.5 ± 1.3 – fold increase in titer (P = 0.008), respectively. A histone gel assay confirmed increased histone acetylation in treated producer cells. Conclusion These results show that SIN retrovectors incorporating strong internal promoters such as CMVIE, are susceptible to transcriptional silencing and that treatment of the producer cells with HDAC inhibitors can overcome this blockade suggesting that histone deacetylation is implicated in the mechanism of transcriptional suppression. PMID:16603064

  16. Nerve growth factor is involved in the supportive effect by bone marrow--derived stromal cells of the factor-dependent human cell line UT-7.

    PubMed

    Auffray, I; Chevalier, S; Froger, J; Izac, B; Vainchenker, W; Gascan, H; Coulombel, L

    1996-09-01

    We previously demonstrated that murine MS-5 and SI/SI4 cell lines induce the proliferation of human factor-dependent UT-7 cells in the absence of normally required human cytokines and also stimulate the differentiation of CD34+/CD38-LTC-ICs. We report in this study that the effect of MS-5 cells on UT-7 cells can be completely explained by the synergistic action of nerve growth factor (NGF) and stem cell factor (SCF) produced by these murine stromal cells. Purified murine NGF was able to support short-term clone formation and long-term growth of UT-7 cells in suspension cultures as efficiently as rhu-granulocyte-macrophage colony-stimulating factor. NGF action was mediated through the TrkA receptor, in which messenger RNA (mRNA) was easily detected in UT-7 cells by Northern blot. MS-5 cells strongly expressed NGF mRNA in Northern blot, and direct implication of MS-5-derived NGF in the induction of UT-7 cells proliferation was demonstrated in inhibition assays with an anti-NGF monoclonal antibody (MoAb) that neutralized by 84% +/- 4.1% (n = 5) UT-7 clone formation. However, NGF did not act alone, and several arguments demonstrated the synergistic action of MS-5-derived SCF: (1) an anti-c-kit partially inhibited UT-7 cells clone formation in coculture assays, (2) SCF and NGF synergized in an H3-TdR incorporation assay, and (3) the stimulatory effect of 10x-concentrated MS-5 supernatant was completely inhibited by an anti-c-kit but not by an anti-NGF, and levels of soluble NGF (1.2 ng/mL) detected by enzyme-linked immunosorbent assay in 10x supernatant of MS-5 cells cultures were below the biologically active concentrations. In contrast, although MS-5 cells also promoted the differentiation of very primitive CD34+/CD38- human stem cells both in colony assays and long-term cultures, we could not incriminate MS-5-derived NGF in the observed effect: an anti-NGF MoAb did not inhibit the synergistic effect of MS-5 cells in colony assays or long-term cultures nor did soluble

  17. Collagen gel droplet-embedded culture drug sensitivity testing in squamous cell carcinoma cell lines derived from human oral cancers: Optimal contact concentrations of cisplatin and fluorouracil

    PubMed Central

    Sakuma, Kaname; Tanaka, Akira; Mataga, Izumi

    2016-01-01

    The collagen gel droplet-embedded culture drug sensitivity test (CD-DST) is an anticancer drug sensitivity test that uses a method of three-dimensional culture of extremely small samples, and it is suited to primary cultures of human cancer cells. It is a useful method for oral squamous cell carcinoma (OSCC), in which the cancer tissues available for testing are limited. However, since the optimal contact concentrations of anticancer drugs have yet to be established in OSCC, CD-DST for detecting drug sensitivities of OSCC is currently performed by applying the optimal contact concentrations for stomach cancer. In the present study, squamous carcinoma cell lines from human oral cancer were used to investigate the optimal contact concentrations of cisplatin (CDDP) and fluorouracil (5-FU) during CD-DST for OSCC. CD-DST was performed in 7 squamous cell carcinoma cell lines derived from human oral cancers (Ca9-22, HSC-3, HSC-4, HO-1-N-1, KON, OSC-19 and SAS) using CDDP (0.15, 0.3, 1.25, 2.5, 5.0 and 10.0 µg/ml) and 5-FU (0.4, 0.9, 1.8, 3.8, 7.5, 15.0 and 30.0 µg/ml), and the optimal contact concentrations were calculated from the clinical response rate of OSCC to single-drug treatment and the in vitro efficacy rate curve. The optimal concentrations were 0.5 µg/ml for CDDP and 0.7 µg/ml for 5-FU. The antitumor efficacy of CDDP at this optimal contact concentration in CD-DST was compared to the antitumor efficacy in the nude mouse method. The T/C values, which were calculated as the ratio of the colony volume of the treatment group and the colony volume of the control group, at the optimal contact concentration of CDDP and of the nude mouse method were almost in agreement (P<0.05) and predicted clinical efficacy, indicating that the calculated optimal contact concentration is valid. Therefore, chemotherapy for OSCC based on anticancer drug sensitivity tests offers patients a greater freedom of choice and is likely to assume a greater importance in the selection of

  18. Positive Selection and Multiple Losses of the LINE-1-Derived L1TD1 Gene in Mammals Suggest a Dual Role in Genome Defense and Pluripotency

    PubMed Central

    Yang, Lei; Neme, Rafik; Wichman, Holly A.; Malik, Harmit S.

    2014-01-01

    Mammalian genomes comprise many active and fossilized retroelements. The obligate requirement for retroelement integration affords host genomes an opportunity to ‘domesticate’ retroelement genes for their own purpose, leading to important innovations in genome defense and placentation. While many such exaptations involve retroviruses, the L1TD1 gene is the only known domesticated gene whose protein-coding sequence is almost entirely derived from a LINE-1 (L1) retroelement. Human L1TD1 has been shown to play an important role in pluripotency maintenance. To investigate how this role was acquired, we traced the origin and evolution of L1TD1. We find that L1TD1 originated in the common ancestor of eutherian mammals, but was lost or pseudogenized multiple times during mammalian evolution. We also find that L1TD1 has evolved under positive selection during primate and mouse evolution, and that one prosimian L1TD1 has ‘replenished’ itself with a more recent L1 ORF1 from the prosimian genome. These data suggest that L1TD1 has been recurrently selected for functional novelty, perhaps for a role in genome defense. L1TD1 loss is associated with L1 extinction in several megabat lineages, but not in sigmodontine rodents. We hypothesize that L1TD1 could have originally evolved for genome defense against L1 elements. Later, L1TD1 may have become incorporated into pluripotency maintenance in some lineages. Our study highlights the role of retroelement gene domestication in fundamental aspects of mammalian biology, and that such domesticated genes can adopt different functions in different lineages. PMID:25211013

  19. Effect of glial cell line-derived neurotrophic factor on behavior and key members of the brain serotonin system in mouse strains genetically predisposed to behavioral disorders.

    PubMed

    Naumenko, Vladimir S; Bazovkina, Daria V; Semenova, Alina A; Tsybko, Anton S; Il'chibaeva, Tatyana V; Kondaurova, Elena M; Popova, Nina K

    2013-12-01

    The effect of glial cell line-derived neurotrophic factor (GDNF) on behavior and on the serotonin (5-HT) system of a mouse strain predisposed to depressive-like behavior, ASC/Icg (Antidepressant Sensitive Cataleptics), in comparison with the parental "nondepressive" CBA/Lac mice was studied. Within 7 days after acute administration, GDNF (800 ng, i.c.v.) decreased cataleptic immobility but increased depressive-like behavioral traits in both investigated mouse strains and produced anxiolytic effects in ASC mice. The expression of the gene encoding the key enzyme for 5-HT biosynthesis in the brain, tryptophan hydroxylase-2 (Tph-2), and 5-HT1A receptor gene in the midbrain as well as 5-HT2A receptor gene in the frontal cortex were increased in GDNF-treated ASC mice. At the same time, GDNF decreased 5-HT1A and 5-HT2A receptor gene expression in the hippocampus of ASC mice. GDNF failed to change Tph2, 5-HT1A , or 5-HT2A receptor mRNA levels in CBA mice as well as 5-HT transporter gene expression and 5-HT1A and 5-HT2A receptor functional activity in both investigated mouse strains. The results show 1) a GDNF-induced increase in the expression of key genes of the brain 5-HT system, Tph2, 5-HT1A , and 5-HT2A receptors, and 2) significant genotype-dependent differences in the 5-HT system response to GDNF treatment. The data suggest that genetically defined cross-talk between neurotrophic factors and the brain 5-HT system underlies the variability in behavioral response to GDNF.

  20. Relationship Between Chronic Tinnitus and Glial Cell Line-Derived Neurotrophic Factor Gene rs3812047, rs1110149, and rs884344 Polymorphisms in a Turkish Population.

    PubMed

    Orenay-Boyacioglu, Seda; Coskunoglu, Aysun; Caki, Zerrin; Cam, Fethi Sirri

    2016-08-01

    Glial cell line-derived neurotrophic factor (GDNF) plays a key role in early development of central auditory pathway and the inner ear. However, the auditory pathway studies of GDNF gene polymorphisms are scarce in the literature, and the studies especially associated with tinnitus are limited. Our study aimed to identify whether GDNF gene polymorphisms play any roles in the pathophysiology of tinnitus by investigating the relationship between tinnitus and GDNF polymorphisms. A total of 52 patients with chronic tinnitus and ages ranging from 18 to 55 were admitted to the Ear-Nose-Throat outpatient clinic of Celal Bayar University Medical Faculty Hospital of Manisa, Turkey and constituted the study group. Another 42 patients of the same age range, without tinnitus symptoms and lacking any systemic disease, were also admitted to the clinic and formed the control group. The tympanometric, audiological, and psychoacoustic assessments of the subjects were performed. Deoxyribonucleic acid samples obtained using venous blood taken for routine inspections were used to investigate GDNF gene polymorphisms (rs884344, rs3812047, and rs1110149) by polymerase chain reaction-based restriction fragment length polymorphism method. No correlation could be detected between GDNF rs884344 and rs3812047 polymorphisms and subjects with tinnitus (p > 0.05). Heterozygosity was significantly lower for GDNF rs1110149 polymorphism in tinnitus subjects compared to the controls (p < 0.05). However, the allele frequencies for all 3 polymorphisms were not significantly different between tinnitus and control groups (p > 0.05). Failure to detect correlations between tinnitus and GDNF gene polymorphisms suggests this may be due to the fact that the GDNF gene has a variable expression pattern in different tissues and pathologies. Therefore, the study should be improved and its scope should be expanded by including a larger group of patients and different tissues to investigate the expression

  1. Intrathecal injection of lentivirus-mediated glial cell line-derived neurotrophic factor RNA interference relieves bone cancer-induced pain in rats.

    PubMed

    Meng, Fu-Fen; Xu, Yang; Dan, Qi-Qin; Wei, La; Deng, Ying-Jie; Liu, Jia; He, Mu; Liu, Wei; Xia, Qing-Jie; Zhou, Fiona H; Wang, Ting-Hua; Wang, Xi-Yan

    2015-04-01

    Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.

  2. Effect of 50 Hz Extremely Low-Frequency Electromagnetic Fields on the DNA Methylation and DNA Methyltransferases in Mouse Spermatocyte-Derived Cell Line GC-2

    PubMed Central

    Liu, Yong; Liu, Wen-bin; Liu, Kai-jun; Ao, Lin; Zhong, Julia Li; Cao, Jia; Liu, Jin-yi

    2015-01-01

    Previous studies have shown that the male reproductive system is one of the most sensitive organs to electromagnetic radiation. However, the biological effects and molecular mechanism are largely unclear. Our study was designed to elucidate the epigenetic effects of 50 Hz ELF-EMF in vitro. Mouse spermatocyte-derived GC-2 cell line was exposed to 50 Hz ELF-EMF (5 min on and 10 min off) at magnetic field intensity of 1 mT, 2 mT, and 3 mT with an intermittent exposure for 72 h. We found that 50 Hz ELF-EMF exposure decreased genome-wide methylation at 1 mT, but global methylation was higher at 3 mT compared with the controls. The expression of DNMT1 and DNMT3b was decreased at 1 mT, and 50 Hz ELF-EMF can increase the expression of DNMT1 and DNMT3b of GC-2 cells at 3 mT. However, 50 Hz ELF-EMF had little influence on the expression of DNMT3a. Then, we established DNA methylation and gene expression profiling and validated some genes with aberrant DNA methylation and expression at different intensity of 50 Hz ELF-EMF. These results suggest that the alterations of genome-wide methylation and DNMTs expression may play an important role in the biological effects of 50 Hz ELF-EMF exposure. PMID:26339596

  3. Repair of DNA-protein cross-links in an excision repair-deficient human cell line and its simian virus 40-transformed derivative

    SciTech Connect

    Gantt, R.; Taylor, W.G.; Camalier, R.F.; Stephens, E.V.

    1984-05-01

    DNA-protein cross-links are induced in mammalian cells by X-rays, ultraviolet light, fluorescent light, and numerous chemical carcinogens. Others have shown that these cross-links are repaired by normal cells but that excision repair-deficient xeroderma pigmentosum (XP) Group A cells, XP12BE, are deficient in repair of these bulky adducts. This paper compares the DNA-protein cross-link repair competency of another XP Group A strain, XP20S, with its more rapidly proliferating simian virus 40-transformed derivative line and with normal human skin fibroblasts. DNA-protein cross-links were induced with 20 microM transplatinum(II)diamminedichloride and assayed by the membrane alkaline elution procedure of Kohn. Treated and untreated cells are lysed on a polycarbonate membrane filter, and the coelution rates of the DNA at pH 12.2 are compared; DNA-protein cross-links retard elution of DNA. The repair competency of XP20S cells for trans-platinum(II)diamminedichloride-induced DNA-protein cross-links was similar to that of XP12BE cells, but the competency of the simian virus 40-transformed XP20S cells was nearly equal to that of normal human skin fibroblasts. These results suggest that either cell cycling compensates for the genetic deficiency present in the nucleotide excision process of XP Group A cells or that a process other than nucleotide excision can repair these lesions; this process requires cell cycling or activation by the virus.

  4. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    PubMed

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  5. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells*

    PubMed Central

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-01-01

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gαi/o inhibitor, but not by NF449, a Gαs inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gαο1 and Gαi3 by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKeyTM assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gαi/o activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gαi/o upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants. PMID:25869129

  6. Diet-induced obesity has neuroprotective effects in murine gastric enteric nervous system: involvement of leptin and glial cell line-derived neurotrophic factor.

    PubMed

    Baudry, Charlotte; Reichardt, François; Marchix, Justine; Bado, André; Schemann, Michael; des Varannes, Stanislas Bruley; Neunlist, Michel; Moriez, Raphaël

    2012-02-01

    Nutritional factors can induce profound neuroplastic changes in the enteric nervous system (ENS), responsible for changes in gastrointestinal (GI) motility. However, long-term effects of a nutritional imbalance leading to obesity, such as Western diet (WD), upon ENS phenotype and control of GI motility remain unknown. Therefore, we investigated the effects of WD-induced obesity (DIO) on ENS phenotype and function as well as factors involved in functional plasticity. Mice were fed with normal diet (ND) or WD for 12 weeks. GI motility was assessed in vivo and ex vivo. Myenteric neurons and glia were analysed with immunohistochemical methods using antibodies against Hu, neuronal nitric oxide synthase (nNOS), Sox-10 and with calcium imaging techniques. Leptin and glial cell line-derived neurotrophic factor (GDNF) were studied using immunohistochemical, biochemical or PCR methods in mice and primary culture of ENS. DIO prevented the age-associated decrease in antral nitrergic neurons observed in ND mice. Nerve stimulation evoked a stronger neuronal Ca(2+) response in WD compared to ND mice. DIO induced an NO-dependent increase in gastric emptying and neuromuscular transmission in the antrum without any change in small intestinal transit. During WD but not ND, a time-dependent increase in leptin and GDNF occurred in the antrum. Finally, we showed that leptin increased GDNF production in the ENS and induced neuroprotective effects mediated in part by GDNF. These results demonstrate that DIO induces neuroplastic changes in the antrum leading to an NO-dependent acceleration of gastric emptying. In addition, DIO induced neuroplasticity in the ENS is likely to involve leptin and GDNF.

  7. Synthesis of a Biotin Derivative of Iberiotoxin: Binding Interactions with Streptavidin and the BK Ca2+-activated K+ Channel Expressed in a Human Cell Line

    PubMed Central

    Bingham, Jon-Paul; Bian, Shumin; Tan, Zhi-Yong; Takacs, Zoltan; Moczydlowski, Edward

    2008-01-01

    Iberiotoxin (IbTx) is a scorpion venom peptide that inhibits BK Ca2+-activated K+ channels with high affinity and specificity. Automated solid phase synthesis was used to prepare a biotin-labeled derivative (IbTx-LC-biotin) of IbTx by substitution of Asp19 of the native 37-residue peptide with N-ε-(d-biotin-6-amidocaproate)-l-lysine. Both IbTx-LC-biotin and its complex with streptavidin (StrAv) block single BK channels from rat skeletal muscle with nanomolar affinity, indicating that the biotin-labeled residue, either alone or in complex with StrAv, does not obstruct the toxin binding interaction with the BK channel. IbTx-LC-biotin exhibits high affinity (KD = 26 nM) and a slow dissociation rate (koff = 5.4 × 10-4 s-1) in a macroscopic blocking assay of whole-cell current of the cloned human BK channel. Titration of IbTx-LC-biotin with StrAv monitored by high performance size exclusion chromatography is consistent with a stoichiometry of two binding sites for IbTx-LC-biotin per StrAv tetramer, indicating that steric interference hinders simultaneous binding of two toxin molecules on each of the two biotin-binding faces of StrAv. In combination with fluorescent conjugates of StrAv or anti-biotin antibody, IbTx-LC-biotin was used to image the surface distribution of BK channels on a transfected cell line. Fluorescence microscopy revealed a patch-like surface distribution of BK channel protein. The results support the feasibility of using IbTx-LC-biotin and similar biotin-tagged K+ channel toxins for diverse applications in cellular neurobiology. PMID:16704206

  8. Sciatic nerve injury in adult rats causes distinct changes in the central projections of sensory neurons expressing different glial cell line-derived neurotrophic factor family receptors

    PubMed Central

    Keast, Janet R.; Forrest, Shelley L.; Osborne, Peregrine B.

    2010-01-01

    Most small unmyelinated neurons in adult rat dorsal ganglia (DRG) express one or more of the co-receptors targeted by glial cell line-derived neurotrophic factor (GDNF), neurturin and artemin (GFRα1, GFRα2 and GFRα3 respectively). The function of these GDNF family ligands (GFLs) is not fully elucidated but recent evidence suggests GFLs could function in sensory neuron regeneration after nerve injury and peripheral nociceptor sensitisation. In this study, we used immunohistochemistry to determine if the DRG neurons targeted by each GFL change after sciatic nerve injury. We compared complete sciatic nerve transection and the chronic constriction model and found the pattern of changes incurred by each injury was broadly similar. In lumbar spinal cord, there was a widespread increase in neuronal GFRα1 immunoreactivity (IR) in the L1-6 dorsal horn. GFRα3-IR also increased but in a more restricted area. In contrast, GFRα2-IR decreased in patches of superficial dorsal horn and this loss was more extensive after transection injury. No change in calcitonin gene-related peptide-IR was detected after either injury. Analysis of double-immunolabelled L5 DRG sections suggested the main effect of injury on GFRα1- and GFRα3-IR was to increase expression in both myelinated and unmyelinated neurons. In contrast, no change in basal expression of GFRα2-IR was detected in DRG by analysis of fluorescence intensity and there was a small but significant reduction in GFRα2-IR neurons. Our results suggest the DRG neuronal populations targeted by GDNF, neurturin or artemin, and the effect of exogenous GFLs could change significantly after a peripheral nerve injury. PMID:20533358

  9. Glial cell line-derived neurotrophic factor (GDNF) protein content in rat skeletal muscle is altered by increased physical activity in vivo and in vitro

    PubMed Central

    McCullough, Monica J.; Peplinski, Nathan G.; Kinnell, Kyle R.; Spitsbergen, John M.

    2010-01-01

    Current evidence suggests that exercise and glial cell line-derived neurotrophic factor (GDNF) independently cause significant morphological changes in the neuromuscular system. The aim of the current study was to determine if increased physical activity regulates GDNF protein content in rat skeletal muscle. Extensor Digitorum Longus (EDL) and Soleus (SOL) hindlimb skeletal muscles were analyzed following 2 weeks of involuntary exercise and 4 hours of field stimulation or stretch in muscle bath preparations. GDNF protein content was measured via ELISA. Two weeks of exercise increased GDNF protein content in SOL as compared to sedentary controls (4.4 ±0.3 pg GDNF/mg tissue and 3.1 ±0.6 pg GDNF/mg tissue, respectively) and decreased GDNF protein content in EDL as compared to controls (1.0 ±0.1 pg GDNF/mg tissue and 2.3 ±0.7 pg GDNF/mg tissue, respectively). GDNF protein content in the EDL decreased following both field stimulation (56% ±18% decrease from controls) and stretch (66% ±10% decrease from controls). SOL responded to field stimulation with a 38% ±7% increase from controls in GDNF protein content, but showed no change following stretch. Pre-treatment with α-bungarotoxin abolished the effects of field stimulation in both muscles and blocked the effect of stretch in EDL. α-bungarotoxin pre-treatment and stretch increased GDNF protein content to 240% ±10% of controls in the SOL. Exposure to carbamylcholine decreased GDNF protein content to 51% ±28% of controls in the EDL but not SOL. These results suggest that GDNF protein content in skeletal muscle may be controlled by stretch, where it may increase GDNF protein content, and membrane depolarization/ACh which acts to decrease GDNF protein content. PMID:21081155

  10. Transgenic expression of human glial cell line-derived neurotrophic factor from integration-deficient lentiviral vectors is neuroprotective in a rodent model of Parkinson's disease.

    PubMed

    Lu-Nguyen, Ngoc B; Broadstock, Martin; Schliesser, Maximilian G; Bartholomae, Cynthia C; von Kalle, Christof; Schmidt, Manfred; Yáñez-Muñoz, Rafael J

    2014-07-01

    Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise for gene therapy of Parkinson's disease (PD). Their main drawback is the risk of insertional mutagenesis. The novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) may offer a significant enhancement in biosafety, but have not been previously tested in a model of a major disease. We have assessed biosafety and transduction efficiency of IDLVs in a rat model of PD, using IPLVs as a reference. Genomic insertion of lentivectors injected into the lesioned striatum was studied by linear amplification-mediated polymerase chain reaction (PCR), followed by deep sequencing and insertion site analysis, demonstrating lack of significant IDLV integration. Reporter gene expression studies showed efficient, long-lived, and transcriptionally targeted expression from IDLVs injected ahead of lesioning in the rat striatum, although at somewhat lower expression levels than from IPLVs. Transgenic human glial cell line-derived neurotrophic factor (hGDNF) expression from IDLVs was used for a long-term investigation of lentivector-mediated, transcriptionally targeted neuroprotection in this PD rat model. Vectors were injected before striatal lesioning, and the results showed improvements in nigral dopaminergic neuron survival and behavioral tests regardless of lentiviral integration proficiency, although they confirmed lower expression levels of hGDNF from IDLVs. These data demonstrate the effectiveness of IDLVs in a model of a major disease and indicate that these vectors could provide long-term PD treatment at low dose, combining efficacy and biosafety for targeted central nervous system applications.

  11. Tricyclic Antidepressant Amitriptyline-induced Glial Cell Line-derived Neurotrophic Factor Production Involves Pertussis Toxin-sensitive Gαi/o Activation in Astroglial Cells.

    PubMed

    Hisaoka-Nakashima, Kazue; Miyano, Kanako; Matsumoto, Chie; Kajitani, Naoto; Abe, Hiromi; Okada-Tsuchioka, Mami; Yokoyama, Akinobu; Uezono, Yasuhito; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2015-05-29

    Further elaborating the mechanism of antidepressants, beyond modulation of monoaminergic neurotransmission, this study sought to elucidate the mechanism of amitriptyline-induced production of glial cell line-derived neurotrophic factor (GDNF) in astroglial cells. Previous studies demonstrated that an amitriptyline-evoked matrix metalloproteinase (MMP)/FGF receptor (FGFR)/FGFR substrate 2α (FRS2α)/ERK cascade is crucial for GDNF production, but how amitriptyline triggers this cascade remains unknown. MMP is activated by intracellular mediators such as G proteins, and this study sought to clarify the involvement of G protein signaling in amitriptyline-evoked GDNF production in rat C6 astroglial cells (C6 cells), primary cultured rat astrocytes, and normal human astrocytes. Amitriptyline-evoked GDNF mRNA expression and release were inhibited by pertussis toxin (PTX), a Gα(i/o) inhibitor, but not by NF449, a Gα(s) inhibitor, or YM-254890, a Gαq inhibitor. The activation of the GDNF production cascade (FGFR/FRS2α/ERK) was also inhibited by PTX. Deletion of Gα(ο1) and Gα(i3) by RNAi demonstrated that these G proteins play important roles in amitriptyline signaling. G protein activation was directly analyzed by electrical impedance-based biosensors (CellKey(TM) assay), using a label-free (without use of fluorescent proteins/probes or radioisotopes) and real time approach. Amitriptyline increased impedance, indicating Gα(i/o) activation that was suppressed by PTX treatment. The impedance evoked by amitriptyline was not affected by inhibitors of the GDNF production cascade. Furthermore, FGF2 treatment did not elicit any effect on impedance, indicating that amitriptyline targets PTX-sensitive Gα(i/o) upstream of the MMP/FGFR/FRS2α/ERK cascade. These results suggest novel targeting for the development of antidepressants.

  12. Glial cell line-derived neurotrophic factor (GDNF) induced migration of spermatogonial cells in vitro via MEK and NF-kB pathways.

    PubMed

    Huleihel, M; Fadlon, E; Abuelhija, A; Piltcher Haber, E; Lunenfeld, E

    2013-01-01

    Glial cell line-derived neurotrophic factor (GDNF) regulates spermatogonial stem cell (SSC) maintenance. In the present study, we examined the levels and the cellular origin of GDNF in mouse testes during age-development, and the capacity of GDNF to induce migration of enriched GFR-α1 positive cells in vitro. The involvement of MAP kinase (MEK) and NF-kB signal pathways were examined. Our results show high levels of GDNF in testicular tissue of one-week-old mice which significantly decreased with age when examined by ELISA, real time PCR (qPCR) and immunofluorescence staining (IF) analysis. GDNF receptor (GFR-α1) expression was similar to GDNF when examined by qPCR analysis. Only Sertoli cell cultures (SCs) from one-week-old mice produced GDNF compared to SCs from older mice. However, peritubular cells from all the examined ages did not produce GDNF. The addition of recombinant GDNF (rGDNF) or supernatant from SCs from one-week-old mice to GFR-α1 positive cells induced their migration in vitro. This effect was significantly reduced by the addition of inhibitors to MEK (PD98059, U0126), NF-kB (PDTC) and IkB protease inhibitor (TPCK). Our results show for the first time the capacity of rGDNF and supernatant from SCs to induce migration of enriched GFR-α1 positive cells, and the possible involvement of MEK, NF-kB and IkB in this process. This study may suggest a novel role for GDNF in the regulation SSC niches and spermatogenesis.

  13. Glial cell line-derived neurotrophic factor modulates the excitability of nociceptive trigeminal ganglion neurons via a paracrine mechanism following inflammation.

    PubMed

    Takeda, Mamoru; Takahashi, Masayuki; Hara, Norifumi; Matsumoto, Shigeji

    2013-02-01

    Previous our report indicated that acute application of glial cell line-derived neurotrophic factor (GDNF) enhances the neuronal excitability of adult rat small-diameter trigeminal ganglion (TRG) neurons, which innervate the facial skin in the absence of neuropathic and inflammatory conditions. This study investigated whether under in vivo conditions, GDNF modulates the excitability of nociceptive Aδ-TRG neurons innervating the facial skin via a paracrine mechanism following inflammation. We used extracellular electrophysiological recording with multibarrel-electrodes in this study. Spontaneous Aδ-TRG neuronal activity was induced in control rats after iontophoretic application of GDNF into the trigeminal ganglia (TRGs). Noxious and non-noxious mechanical stimuli evoked Aδ-TRG neuronal firing rate were significantly increased by iontophoretic application of GDNF. The mean mechanical threshold of nociceptive TRG neurons was significantly decreased by GDNF application. The increased discharge frequency and decreased mechanical threshold induced by GDNF were antagonized by application of the protein tyrosine kinase inhibitor, K252b. The number of Aδ-TRG neurons with spontaneous firings and their firing rates in rats with inflammation induced by Complete Freund's Adjuvant were significantly higher than control rats. The firing rates of Aδ-TRG spontaneous neuronal activity were significantly decreased by iontophoretic application of K252b in inflamed rats. K252b also inhibited Aδ-TRG neuron activity evoked by mechanical stimulation in inflamed rats. These results suggest that in vivo GDNF enhances the excitability of nociceptive Aδ-TRG neurons via a paracrine mechanism within TRGs following inflammation. GDNF paracrine mechanism could be important as a therapeutic target for trigeminal inflammatory hyperalgesia.

  14. 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

    PubMed Central

    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. PMID:27191603

  15. Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

    SciTech Connect

    Sebastian, Katrin; Ott, Hagen; Zwadlo-Klarwasser, Gabriele; Skazik-Voogt, Claudia; Marquardt, Yvonne; Czaja, Katharina; Merk, Hans F.; Baron, Jens Malte

    2012-08-01

    Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.

  16. Tricyclic antidepressant amitriptyline activates fibroblast growth factor receptor signaling in glial cells: involvement in glial cell line-derived neurotrophic factor production.

    PubMed

    Hisaoka, Kazue; Tsuchioka, Mami; Yano, Ryoya; Maeda, Natsuko; Kajitani, Naoto; Morioka, Norimitsu; Nakata, Yoshihiro; Takebayashi, Minoru

    2011-06-17

    Recently, both clinical and animal studies demonstrated neuronal and glial plasticity to be important for the therapeutic action of antidepressants. Antidepressants increase glial cell line-derived neurotrophic factor (GDNF) production through monoamine-independent protein-tyrosine kinase, extracellular signal-regulated kinase (ERK), and cAMP responsive element-binding protein (CREB) activation in glial cells (Hisaoka, K., Takebayashi, M., Tsuchioka, M., Maeda, N., Nakata, Y., and Yamawaki, S. (2007) J. Pharmacol. Exp. Ther. 321, 148-157; Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Brain Res. 1196, 53-58). This study clarifies the type of tyrosine kinase and mechanism of antidepressant-induced GDNF production in C6 glioma cells and normal human astrocytes. The amitriptyline (a tricyclic antidepressant)-induced ERK activation was specifically and completely inhibited by fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitors and siRNA for FGFR1 and -2. Treatment with amitriptyline or several different classes of antidepressants, but not non-antidepressants, acutely increased the phosphorylation of FGFRs and FGFR substrate 2α (FRS2α). Amitriptyline-induced CREB phosphorylation and GDNF production were blocked by FGFR-tyrosine kinase inhibitors. Therefore, antidepressants activate the FGFR/FRS2α/ERK/CREB signaling cascade, thus resulting in GDNF production. Furthermore, we attempted to elucidate how antidepressants activate FGFR signaling. The effect of amitriptyline was inhibited by heparin, non-permeant FGF-2 neutralizing antibodies, and matrix metalloproteinase (MMP) inhibitors. Serotonin (5-HT) also increased GDNF production through FGFR2 (Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., and Nakata, Y. (2008) J. Neurochem. 106, 244-257); however, the effect of 5-HT was not inhibited by heparin and MMP inhibitors. These results suggest that amitriptyline-induced FGFR activation might occur through an extracellular pathway

  17. Genetic Diversity and Phenotypic Variation in an Introgression Line Population Derived from an Interspecific Cross between Oryza glaberrima and Oryza sativa

    PubMed Central

    Nassirou, Tondi Yacouba; Zhou, Wei; Yin, Yilong; Dong, Xilong; Rao, Quanqin; Shi, Han; Zhao, Wubin; Efisue, Andrew; Jin, Deming

    2016-01-01

    The introduction of closely related species genomic fragments is an effective way to enrich genetic diversity and creates new germplasms in crops. Here, we studied the genetic diversity of an introgression line (IL) population composed of 106 ILs derived from an interspecific tetra cross between O. glaberrima and O. sativa (RAM3/Jin23B//Jin23B///YuetaiB). The proportion of O. glaberrima genome (PGG) in the ILs ranged from 0.3% to 36.7%, with an average value of 12.32% which is close to the theoretically expected proportion. A total of 250 polymorphic alleles were amplified by 21 AFLP primer combinations with an average of 12 alleles per primer. Population structure analysis revealed that the IL population can be divided into four genetically distinct subpopulations. Both principal component analysis and neighbor-joining tree analysis showed that ILs with a higher PGG displayed greater genetic diversity. Canonical discriminant analysis identified six phenotypic traits (plant height, yield per plant, filled grain percentage, panicle length, panicle number and days to flowering) as the main discriminatory traits among the ILs and between the subpopulations and showed significant phenotypic distances between subpopulations. The effects of PGG on phenotypic traits in the ILs were estimated using a linear admixed model, which showed a significant positive effect on grain yield per plant (0.286±0.117), plant height (0.418 ± 0.132), panicle length (0.663 ± 0.107), and spikelet number per panicle (0.339 ± 0.128), and a significant negative effect on filled grain percentage (-0.267 ± 0.123) and days to flowering (-0.324 ± 0.075). We found that an intermediate range (10% − 20%) of PGG was more effective for producing ILs with favorable integrated agronomic traits. Our results confirm that construction of IL population carrying O. glaberrima genomic fragments could be an effective approach to increase the genetic diversity of O. sativa genome and an appropriate level

  18. Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase

    PubMed Central

    Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

    2015-01-01

    The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT. PMID:26496927

  19. Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.

    PubMed

    Goto, Hiroaki; Naruto, Takuya; Tanoshima, Reo; Kato, Hiromi; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Fujii, Hisaki; Yokota, Shumpei; Komine, Hiromi

    2009-10-01

    Sensitivity to 10 anticancer drugs was evaluated in 6 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. Authenticity of newly established cell lines was confirmed by genomic fingerprinting. The line YCUB-5R established at relapse was more resistant to 4-hydroperoxy-cyclophosphamide, cytarabine, L-asparaginase, topotecan, fludarabine, and etoposide than YCUB-5 from the same patient at diagnosis. Of the drugs tested, etoposide and SN-38 (irinotecan) showed highest efficacy in the panel, with 50% growth inhibition at 0.22-1.8 microg/ml and 0.57-3.6 ng/ml, respectively. This cell line panel offers an in vitro model for the development of new therapies for childhood BCP-ALL.

  20. cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

    PubMed

    Smith, A N; Wardle, C J; Winpenny, J P; Verdon, B; Gray, M A; Argent, B E; Harris, A

    1995-06-09

    Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

  1. Relative coronal abundances derived from X-ray observations 3: The effect of cascades on the relative intensity of Fe (XVII) line fluxes, and a revised iron abundance

    NASA Technical Reports Server (NTRS)

    Walker, A. B. C., Jr.; Rugge, H. R.; Weiss, K.

    1974-01-01

    Permitted lines in the optically thin coronal X-ray spectrum were analyzed to find the distribution of coronal material, as a function of temperature, without special assumptions concerning coronal conditions. The resonance lines of N, O, Ne, Na, Mg, Al, Si, S, and Ar which dominate the quiet coronal spectrum below 25A were observed. Coronal models were constructed and the relative abundances of these elements were determined. The intensity in the lines of the 2p-3d transitions near 15A was used in conjunction with these coronal models, with the assumption of coronal excitation, to determine the Fe XVII abundance. The relative intensities of the 2p-3d Fe XVII lines observed in the corona agreed with theoretical prediction. Using a more complete theoretical model, and higher resolution observations, a revised calculation of iron abundance relative to hydrogen of 0.000026 was made.

  2. Synthesis and cytotoxic activities of some 2-arylnaphtho[2,3-d]oxazole-4,9-dione derivatives on androgen-dependent (LNCaP) and androgen-independent (PC3) human prostate cancer cell lines.

    PubMed

    Brandy, Yakini; Ononiwu, Innocent; Adedeji, Dolapo; Williams, Vonetta; Mouamba, Claudia; Kanaan, Yasmine; Copeland, Robert L; Wright, Dwayne A; Butcher, Ray J; Denmeade, Samuel R; Bakare, Oladapo

    2012-08-01

    The synthesis of five 2-arylnaphtho[2,3-d]oxazole-4,9-dione derivatives was accomplished by refluxing 2-amino-3-bromo-1,4-naphthoquinone with appropriate benzoyl chloride analogs at elevated temperatures. In vitro anticancer evaluation of these compounds was performed on androgen-dependent, LNCaP, and androgen-independent, PC3, human prostate cancer cell lines. In general, these compounds displayed slightly stronger cytotoxicity on the androgen-dependent LNCaP than on the androgen-independent PC3 prostate cancer cell lines. The meta-substituted 2-(3-Chloro-phenyl)-naphtho[2,3-d]oxazole-4,9-dione (10) appear to display the best cytotoxicity on both cell lines with an IC(50) of 0.03 μM on LNCaP and 0.08 μM on PC3 after 5 days of exposure.

  3. Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19.

    PubMed

    Talbot, N C; Caperna, T J; Garrett, W M

    2013-01-01

    Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent

  4. Murine myeloid cell lines derived by in vitro infection with recombinant c-myb retroviruses express myb from rearranged vector proviruses.

    PubMed Central

    Gonda, T J; Ramsay, R G; Johnson, G R

    1989-01-01

    To date, cellular transformation in vitro by the myb oncogene has been described for avian haemopoietic cells only. In order to exploit the well-characterized murine haemopoietic system to study transformation by myb, we have infected fetal liver cells with retroviral vectors carrying cDNAs that encode either complete or carboxy-terminally truncated c-myb proteins. We describe four cell lines which, despite our ability to efficiently infect haemopoietic target cells, were generated at low frequency. This was due, as least in part, to the requirement for a rearrangement within the vector that allowed expression of myb sequences. Three of the lines express a truncated myb protein while the fourth apparently expresses a normal c-myb protein, and thus constitutes an exception to the general association of truncation with transformation by myb. All four cell lines resemble immature cells of the myelomonocytic lineage and are dependent on colony-stimulating factors (CSFs) for their growth in vitro. One representative line could be converted to CSF-independence by infection with either Abelson murine leukaemia virus or a recombinant granulocyte-macrophage-CSF-encoding retrovirus; unlike the parental line, the resultant sublines were highly tumorigenic when injected into syngeneic mice. Images PMID:2670561

  5. Short-term selection for acute ethanol tolerance and sensitization from an F2 population derived from the high and low alcohol-sensitive selectively bred rat lines.

    PubMed

    Radcliffe, Richard A; Bludeau, Pequita; Deng, Xin-Sheng; Erwin, V Gene; Deitrich, Richard A

    2007-12-01

    Previous studies have identified quantitative trait loci (QTL) in the inbred high and low alcohol-sensitive rat (IHAS1 and ILAS1) strains. The original development of the strains involved selection for ethanol sensitivity based on duration of the loss of the righting reflex (LORR) after a standard dose of ethanol. This paper confirms some of these QTL using a short-term selection procedure based on the difference between the blood ethanol level at LORR and regain of the righting response. An F(2) population of rats was developed by a reciprocal cross of IHAS1 and ILAS1 rats. Selection for five generations was carried out using delta-blood ethanol concentration (dBEC) as the selection trait, where dBEC=BECLR (BEC at loss of righting reflex)-BECRR (BEC at regain of righting reflex). The lines were labeled tolerant (TOL) or sensitive (SENS). Approximately one-third of the offspring for each generation in each line were genotyped using DNA markers that had been previously found to be linked to QTL on chromosomes 1, 2, 5, 12, and 13. By the fifth generation of selection, the lines showed a very large difference in dBEC, BECRR, and duration of LORR; BECLR showed little segregation during the selection, and latency to lose the righting reflex showed none. IHAS allele frequency increased in the SENS line for markers on chromosomes 1, 5, 12, and 13 while ILAS allele frequency increased in the TOL line. These results were in good agreement with the two previous QTL studies. On chromosome 2, the selection resulted in an accumulation of ILAS alleles in both lines. This study provides independent confirmation of the location of QTL on chromosomes 1, 5, 12, and 13 for ethanol sensitivity. It also suggests that genetic differences in duration of LORR are mediated primarily by the dBEC phenotype.

  6. Evaluation of Exotically-Derived Soybean Breeding Lines for Seed Yield, Germination, Damage, and Composition under Dryland Production in the Midsouthern USA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although the Early Soybean Production System (ESPS) in the Midsouthern USA increased seed yield under irrigated and non-irrigated conditions, heat stress and drought still lead to poor seed quality in heat sensitive soybean cultivars. Our goal was to identify breeding lines that possess high germin...

  7. Identification of glyceollin metabolites derived from conjugation with glutathione and glucuronic acid in rats by on-line liquid chromatography-electrospray ionization tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glyceollin-related metabolites produced in rats following oral glyceollin administration were screened and identified by precursor and product ion scanning using liquid chromatography, coupled on-line with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), to identify all glyceollin me...

  8. Establishment and characterization of a mid-kidney cell line derived from golden pompano Trachinotus ovatus, a new cell model for virus pathogenesis and toxicology studies.

    PubMed

    Zhou, Lingli; Li, Pengfei; Liu, Jiaxin; Ni, Songwei; Yu, Yepin; Yang, Min; Wei, Shina; Qin, Qiwei

    2016-12-15

    Golden pompano Trachinotus ovatus, a popularly cultured and commercially important marine fish worldwide, has been recognized as a promising candidate for mariculture. However, outbreaks of infectious bacterial or viral diseases and environmental deterioration have led to great economic losses in T. ovatus aquaculture recently. In our research, we established a new mid-kidney cell line, designated as TOK, from golden pompano, T. ovatus. The optimized growth temperature and working concentration of fetal bovine serum (FBS) were 28°C and 10-20%, respectively. Foreign genes could express well in TOK cells. The modal number of TOK cells was 54. The TOK cells were susceptive to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), and the virus could propagate in cells. Propagation was verified by qRT-PCR, and virions were observed under electron microscopy. Cytotoxicity analysis revealed that TOK cells were sensitive to different concentrations of extracellular products (ECPs) from Vibrio alginolyticus and V. anguillarum. Moreover, heavy metals (Cd, Cu, and Hg) also showed dose-dependent cytotoxicity to the TOK cell line. We established a mid-kidney cell line from T. ovatus which could be applied to cytotoxicity assays of heavy metals. The newly established TOK cell line possesses great application potential in genetic manipulation, virus-host interaction studies, and toxicity assays of bacterial extracellular products and heavy metals.

  9. Cross-species evaluation of molecular target sequence and structural conservation as a line of evidence for identification of susceptible taxa to inform derivation of aquatic life criteria

    EPA Science Inventory

    The 1985 U.S. Environmental Protection Agency (EPA) Guidelines for Deriving Aquatic Life Criteria (ALC) require acute and chronic toxicity testing with a fixed list of taxa that cover aquatic organisms from vertebrates, invertebrates, and plants. In considering Guideline revision...

  10. Ecofriendly in-line process monitoring: a case study. Anthracene photodegradation in the presence of refuse-derived soluble bio-organics.

    PubMed

    Bianco Prevot, A; Avetta, P; Fabbri, D; Montoneri, E; Morales-Rubio, A; de la Guardia, M

    2012-08-01

    Photodegradation of anthracene has been studied in aqueous solutions containing soluble bio-organic substances isolated from urban refuse. To perform a preliminary rapid feasibility study of this process while reducing the amount of analytical effort and reagents, an experimental set-up was developed comprising a Teflon coil surrounding a UV-lamp and coupled with an in-line spectrofluorimeter. In this fashion only few millilitres of solution are needed to study the degradation process. Furthermore, the in-line spectroscopic approach enables monitoring of the process without consumption of reagents. Additional studies by liquid chromatography and use of toxicity tests clearly indicated that the apparent inhibition effect of bio-organic compounds on anthracene degradation is not relevant. The results imply that urban refuse may be used as an auxiliary in the recovery of polycyclic aromatic hydrocarbons from contaminated soil by washing, without deleterious effects on the photodegradation of anthracene and other aromatic pollutants.

  11. Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

    SciTech Connect

    Li, Xin-xing; Wang, Jian; Wang, Hao-lu; Wang, Wei; Yin, Xiao-bin; Li, Qi-wei; Chen, Yu-ying; Yi, Jing

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.

  12. Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that were derived from nasopharyngeal carcinomas

    SciTech Connect

    Glaser, R.; Zhang, Haizhang ); Yao, Kaitai; Zhu, Hecheng; Wang, Fuxi; Li, Guiyuan; Wen, Dongseng; Li, Yingping )

    1989-12-01

    Two epithelia tumor cell lines were established from biopsy specimens of nasopharyngeal carcinomas (NPC). The specimens were taken from poorly differentiated squamous cell carcinomas of the nasopharynx. The tissues were prepared for cell culture and eventually two continuous epithelia cell lines were obtained and designated HONE-1 and HNE-1. Light and electron microscopic examination of these two cell lines demonstrated cells with an epithelial morphology including the presence of desmosomes. It was found that early-passage uncloned HNE-1 cells (passage 23) could be superinfected with B95-8 and NPC-EBV isolates as demonstrated by the induction of Epstein-Barr virus (EBV)-specific early antigen(s) in a small percentage of the cells; HONE-1 cells could also be superinfected with EBV. Southern blot analysis detected EBV DNA in samples from uncloned HNE-1 cells at passages 12, 17, 21, 27, and 35. However, by passage 45, EBV DNA could no longer be detected in HNE-1 cells by Southern blot analysis. The EBV genome was detected in parental HONE-1 cells at subculture 9 and in clone 40 cells up to passage 40 thus far. The data suggest that EBV genome-positive HNE-1 and HONE-1 cells were lost as the cells were cultivated in vitro and that cloning the cells at an early passage level may be critical in maintaining EBV genome-positive epithelial NPC cells. These EBV genome-positive epithelia NPC cell lines will be useful for studying the association of EBV and NPC.

  13. An anthraquinone derivative from Luffa acutangula induces apoptosis in human lung cancer cell line NCI-H460 through p53-dependent pathway.

    PubMed

    Vanajothi, Ramar; Srinivasan, Pappu

    2016-01-01

    The current study was designed to evaluate the in vitro antiproliferative activity of 1,8-dihydroxy-4-methylanthracene-9,10-dione (DHMA) isolated from the Luffa acutangula against human non-small cell lung cancer cell line (NCI-H460). Induction of apoptosis and reactive oxygen species (ROS) generation was determined through fluorescence microscopic technique. Quantitative real-time PCR and western blotting analysis was carried out to detect the expression of pro-apoptotic (p53, p21, caspase-3, Bax, GADD45A, and ATM) and anti-apoptotic (NF-κB) proteins in NCI-H460 cell line. In silico studies also performed to predict the binding mechanism of DHMA with MDM2-p53 protein. The DHMA inhibited the cell viability of NCI-H460 cells in a dose-dependent manner with an IC(50) of about 50 µg/ml. It significantly reduced cell viability correlated with induction of apoptosis, which was associated with ROS generation. The apoptotic cell death was further confirmed through dual staining and DNA fragmentation assay. DHMA significantly increased the expression of anti-apoptotic protein such as p53, p21, Bax, and caspase-3 but downregulated the expression of NF-κB in NCI-H460 cell line. In silico studies demonstrate that DHMA formed hydrogen bond interaction with key residues Trp26, Phe55 and Lys24 by which it disrupt the binding of p53 with MDM2 receptor. These findings suggested that DHMA induces apoptosis in NCI-H460 via a p53-dependent pathway. This the first study on cytotoxic and apoptosis inducing activity of DHMA from L. acutangula against NCI-H460 cell line. Therefore, DHMA has therapeutic potential for lung cancer treatment.

  14. Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna.

    PubMed

    Bollinedi, Haritha; S, Gopala Krishnan; Prabhu, Kumble Vinod; Singh, Nagendra Kumar; Mishra, Sushma; Khurana, Jitendra P; Singh, Ashok Kumar

    2017-01-01

    Homozygous Golden Rice lines developed in the background of Swarna through marker assisted backcross breeding (MABB) using transgenic GR2-R1 event as a donor for the provitamin A trait have high levels of provitamin A (up to 20 ppm) but are dwarf with pale green leaves and drastically reduced panicle size, grain number and yield as compared to the recurrent parent, Swarna. In this study, we carried out detailed morphological, biochemical and molecular characterization of these lines in a quest to identify the probable reasons for their abnormal phenotype. Nucleotide blast analysis with the primer sequences used to amplify the transgene revealed that the integration of transgene disrupted the native OsAux1 gene, which codes for an auxin transmembrane transporter protein. Real time expression analysis of the transgenes (ZmPsy and CrtI) driven by endosperm-specific promoter revealed the leaky expression of the transgene in the vegetative tissues. We propose that the disruption of OsAux1 disturbed the fine balance of plant growth regulators viz., auxins, gibberellic acid and abscisic acid, leading to the abnormalities in the growth and development of the lines homozygous for the transgene. The study demonstrates the conserved roles of OsAux1 gene in rice and Arabidopsis.

  15. Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna

    PubMed Central

    Bollinedi, Haritha; S., Gopala Krishnan; Prabhu, Kumble Vinod; Singh, Nagendra Kumar; Mishra, Sushma; Khurana, Jitendra P.; Singh, Ashok Kumar

    2017-01-01

    Homozygous Golden Rice lines developed in the background of Swarna through marker assisted backcross breeding (MABB) using transgenic GR2-R1 event as a donor for the provitamin A trait have high levels of provitamin A (up to 20 ppm) but are dwarf with pale green leaves and drastically reduced panicle size, grain number and yield as compared to the recurrent parent, Swarna. In this study, we carried out detailed morphological, biochemical and molecular characterization of these lines in a quest to identify the probable reasons for their abnormal phenotype. Nucleotide blast analysis with the primer sequences used to amplify the transgene revealed that the integration of transgene disrupted the native OsAux1 gene, which codes for an auxin transmembrane transporter protein. Real time expression analysis of the transgenes (ZmPsy and CrtI) driven by endosperm-specific promoter revealed the leaky expression of the transgene in the vegetative tissues. We propose that the disruption of OsAux1 disturbed the fine balance of plant growth regulators viz., auxins, gibberellic acid and abscisic acid, leading to the abnormalities in the growth and development of the lines homozygous for the transgene. The study demonstrates the conserved roles of OsAux1 gene in rice and Arabidopsis. PMID:28068433

  16. Visual exploratory analysis of integrated chromosome 19 proteomic data derived from glioma cancer stem-cell lines based on novel nonlinear dimensional data reduction techniques

    NASA Astrophysics Data System (ADS)

    Lespinats, Sylvain; Pinker-Domenig, Katja; Meyer-Bäse, Uwe; Meyer-Bäse, Anke

    2015-05-01

    Chromosome 19 is known to be linked to neurodegeneration and many cancers. Glioma-derived cancer stem cells (GSCs) are tumor-initiating cells and may be refractory to radiation and chemotherapy and thus have important implications for tumor biology and therapeutics. The analysis and interpretation of large proteomic data sets requires the development of new data mining and visualization approaches. Traditional techniques are insufficient to interpret and visualize these resulting experimental data. The emphasis of this paper lies in the presentation of novel approaches for the visualization, clustering and projection representation to unveil hidden data structures relevant for the accurate interpretation of biological experiments. These qualitative and quantitative methods are applied to the proteomic analysis of data sets derived from the GSCs. The achieved clustering and visualization results provide a more detailed insight into the expression patterns for chromosome 19 proteins.

  17. The Hexane Fraction of Bursera microphylla A Gray Induces p21-Mediated Antiproliferative and Proapoptotic Effects in Human Cancer-Derived Cell Lines.

    PubMed

    Adorisio, Sabrina; Fierabracci, Alessandra; Gigliarelli, Giulia; Muscari, Isabella; Cannarile, Lorenza; Liberati, Anna Marina; Marcotullio, Maria Carla; Riccardi, Carlo; Curini, Massimo; Robles Zepeda, Ramon Enrique; Delfino, Domenico V

    2017-01-01

    Bursera microphylla (BM), one of the common elephant trees, is widely distributed in the Sonoran desert in Mexico. The Seri ethnic group in the Sonoran desert uses BM as an anti-inflammatory and painkiller drug for the treatment of sore throat, herpes labialis, abscessed tooth, and wound healing. Dried stems and leaves of BM are used in a tea to relieve painful urination and to stimulate bronchial secretion. Furthermore, BM is used for fighting venereal diseases. To investigate the effects of the hexane fraction of resin methanol extract (BM-H) on cell growth, the acute myeloid cell line (OCI-AML3) was treated with 250, 25, or 2.5 µg/mL of BM-H. The first 2 concentrations were able to significantly decrease OCI-AML3 cell number. This reduced cell number was associated with decreased S-phase, blockade of G2/M phase of the cell cycle, and increased cell death. Similar results were obtained on all tested tumor cell lines of different origins. We found that blockade of the cell cycle was a result of upregulation of p21 protein in a p53-independent way. Increase of p21 was possibly a result of upstream upregulation of p-ERK (which stabilizes p21 protein) and downregulation of p-38 (which promotes its degradation). Regarding cell death, activation of caspase-3, but not of caspase-8 or -9, was detectable after BM-H treatment. In conclusion, these data suggest that BM-H inhibited proliferation of cell lines mainly by a p21-dependent, p53-independent mechanism and promoted apoptosis through activation of caspase-3 but not caspase-8 or -9.

  18. Inhibition of Oncogenic Transcription Factor REL by the Natural Product Derivative Calafianin Monomer 101 Induces Proliferation Arrest and Apoptosis in Human B-Lymphoma Cell Lines

    PubMed Central

    Yeo, Alan T.; Chennamadhavuni, Spandan; Whitty, Adrian; Porco, John A.; Gilmore, Thomas D.

    2016-01-01

    Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells. PMID:25915462

  19. Differential binding avidities of human IgM for staphylococcal protein A derive from specific germ-line VH3 gene usage.

    PubMed

    Hakoda, M; Kamatani, N; Hayashimoto-Kurumada, S; Silverman, G J; Yamanaka, H; Terai, C; Kashiwazaki, S

    1996-10-01

    Human IgM that express the variable region genes of the VH3 family bind staphylococcal protein A (SPA). We previously reported that the SPA-binding IgM can be divided into two groups based on the differential binding avidities for solid-phase SPA. To study the molecular basis for these differences, we cloned B cells from human blood by EBV transformation. The nucleotide sequences of the expressed Ig heavy chain genes were determined on 20 B cell clones that produce SPA-binding IgM. The germ-line VH3 gene usage in IgM with high avidities for SPA were distinct from the germ-line VH3 genes used in IgM with low avidities for SPA. There was no correlation in the usage of D or JH genes or in the usage of light chains in IgM according to the SPA binding avidity. These results suggest that the differential binding avidities for SPA are at least partly due to specific germ-line VH3 gene usage. An investigation of direct binding of SPA to the synthetic peptides corresponding to the portions of the variable regions of SPA-binding and non-SPA-binding IgM showed that the peptides corresponding to the VH3 family specific framework region 3 sequences had significant SPA binding capacities, while the peptides corresponding to the other subdomains and those corresponding to framework region 3 of the reported VH3 sequences from non-SPA-binding IgM showed little or no binding. It is of interest that the Ig-framework region 3 subdomain corresponds to the fourth hypervariable region, which in the TCR-beta chain has been implicated as a critical site for T cell superantigen binding.

  20. Hydroxynaphthoquinone Metal Complexes as Antitumor Agents X: Synthesis, Structure, Spectroscopy and In Vitro Antitumor Activity of 3-Methyl-Phenylazo Lawsone Derivatives and Their Metal Complexes Against Human Breast Cancer Cell Line MCF-7

    PubMed Central

    Gokhale, Nikhil; Newton, Chris; Pritchard, Robin

    2000-01-01

    The C-3 substituted phenylazo derivatives of lawsone (2-hydroxy-l,4 p-naphthoquinone, III) were synthesized and characterized. The X-ray crystal structure was determined for the ligand 3-(3′-methyl phenylazo) lawsone. The copper complexes of these derivatives were found to possess 1:2 metal stoichiometry and square planar geometries with intermolecular stackings, resulting in antiferromagnetic exchange interactions. The in vitro activity of all the synthesized compounds was examined against human breast cancer cell-line, MCF-7, which revealed enhanced activities for the metal complexes, the highest activity being observed for the copper compound of 3-(3′-methyl phenylazo) lawsone. PMID:18475934

  1. Soy promotes juvenile granulosa cell tumor development in mice and in the human granulosa cell tumor-derived COV434 cell line.

    PubMed

    Mansouri-Attia, Nadéra; James, Rebecca; Ligon, Alysse; Li, Xiaohui; Pangas, Stephanie A

    2014-10-01

    Soy attracts attention for its health benefits, such as lowering cholesterol or preventing breast and colon cancer. Soybeans contain isoflavones, which act as phytoestrogens. Even though isoflavones have beneficial health effects, a role for isoflavones in the initiation and progression of diseases including cancer is becoming increasingly recognized. While data from rodent studies suggest that neonatal exposure to genistein (the predominant isoflavone in soy) disrupts normal reproductive function, its role in ovarian cancers, particularly granulosa cell tumors (GCT), is largely unknown. Our study aimed to define the contribution of a soy diet in GCT development using a genetically modified mouse model for juvenile GCTs (JGCT; Smad1 Smad5 conditional double knockout mice) as well as a human JGCT cell line (COV434). While dietary soy cannot initiate JGCT development in mice, we show that it has dramatic effects on GCT growth and tumor progression compared to a soy-free diet. Loss of Smad1 and Smad5 alters estrogen receptor alpha (Esr1) expression in granulosa cells, perhaps sensitizing the cells to the effects of genistein. In addition, we found that genistein modulates estrogen receptor expression in the human JGCT cell line and positively promotes cell growth in part by suppressing caspase-dependent apoptosis. Combined, our work suggests that dietary soy consumption has deleterious effects on GCT development.

  2. E-configuration structures of EPA and DHA derived from Euphausia superba and their significant inhibitive effects on growth of human cancer cell lines in vitro.

    PubMed

    Zheng, Weilong; Wang, Xudong; Cao, Wenjing; Yang, Bowen; Mu, Ying; Dong, Yuesheng; Xiu, Zhilong

    2017-02-01

    Many bioactive components such as poly-unsaturated fatty acids (e.g. EPA and DHA), phospholipids and astaxanthin are known in Antarctic krill (Euphausia superba) oil. The krill DHA and EPA are generally considered to be similar to natural ones. However, two chemical compounds which were separated from Antarctic krill oil and identified as EPA and DHA by HRESIMS and NMR acted much more effective inhibitive activities on growth of several cell lines (U937, K562, SMMC-7721, PC-3, MDA-MB-231, HL60 and MCF-7) than those from sturgeon liver and commercial fish oil. Taking MCF-7 as an example, the IC50 values of Antarctic krill EPA and DHA were 14.01 and 19.94μM,while the IC50 values of sturgeon liver and commercial fish EPA and DHA were 81.45, 73.13, 82.11 and 75.31μM, respectively. Raman spectra revealed that the Antarctic krill EPA and DHA have E-configuration structures, which were different from those in commercial fish oil. Additionally, the Antarctic krill EPA and DHA had no effects on human normal liver cell line HL7702. These results indicated that the Antarctic krill E-EPA and E-DHA had a great prospect in cancer therapy.

  3. Establishment of a new OSCC cell line derived from OLK and identification of malignant transformation-related proteins by differential proteomics approach

    PubMed Central

    Dong, Yan; Zhao, Qun; Ma, Xiaoyan; Ma, Guowu; Liu, Caiyun; Chen, Zhuwen; Yu, Liyuan; Liu, Xuefeng; Zhang, Yanguang; Shao, Shujuan; Xiao, Jing; Li, Jia; Zhang, Weimin; Fu, Ming; Dong, Lijia; Yang, Xiandong; Guo, Xu; Xue, Liyan; Fang, Fei; Zhan, Qimin; Zhang, Lihua

    2015-01-01

    Oral squamous cell carcinoma (OSCC) is usually preceded by the oral premalignant lesions, mainly oral leukoplakia (OLK) after repeated insults of carcinogens, tobacco. B(a)P and DMBA are key carcinogens in tobacco smoke. In the present study, for the first time we established the cancerous cell line OSCC-BD induced by B(a)P/DMBA mixture and transformed from dysplastic oral leukoplakia cell line DOK. Cell morphology, proliferation ability, migration ability, colony formation, and tumorigenicity were studied and confirmed the malignant characteristics of OSCC-BD cells. We further identified the differential proteins between DOK and OSCC-BD cells by stable isotope dimethyl labeling based quantitative proteomic method, which showed 18 proteins up-regulated and 16 proteins down-regulated with RSD < 8%. Differential proteins are mainly related to cell cycle, cell proliferation, DNA replication, RNA splicing and apoptosis. Abberant binding function, catalysis activity and transportor activity of differential proteins might contribute to the malignant transformation of OLK. Of the 34 identified differential proteins with RSD < 8%, 13 novel cancer-related proteins were reported in the present study. This study might provide a new insight into the mechanism of OLK malignant transformation and the potent biomarkers for early diagnosis, meanwhile further facilitate the application of the quantification proteomics to carcinogenesis research. PMID:26234610

  4. Soy Promotes Juvenile Granulosa Cell Tumor Development in Mice and in the Human Granulosa Cell Tumor-Derived COV434 Cell Line1

    PubMed Central

    Mansouri-Attia, Nadéra; James, Rebecca; Ligon, Alysse; Li, Xiaohui; Pangas, Stephanie A.

    2014-01-01

    ABSTRACT Soy attracts attention for its health benefits, such as lowering cholesterol or preventing breast and colon cancer. Soybeans contain isoflavones, which act as phytoestrogens. Even though isoflavones have beneficial health effects, a role for isoflavones in the initiation and progression of diseases including cancer is becoming increasingly recognized. While data from rodent studies suggest that neonatal exposure to genistein (the predominant isoflavone in soy) disrupts normal reproductive function, its role in ovarian cancers, particularly granulosa cell tumors (GCT), is largely unknown. Our study aimed to define the contribution of a soy diet in GCT development using a genetically modified mouse model for juvenile GCTs (JGCT; Smad1 Smad5 conditional double knockout mice) as well as a human JGCT cell line (COV434). While dietary soy cannot initiate JGCT development in mice, we show that it has dramatic effects on GCT growth and tumor progression compared to a soy-free diet. Loss of Smad1 and Smad5 alters estrogen receptor alpha (Esr1) expression in granulosa cells, perhaps sensitizing the cells to the effects of genistein. In addition, we found that genistein modulates estrogen receptor expression in the human JGCT cell line and positively promotes cell growth in part by suppressing caspase-dependent apoptosis. Combined, our work suggests that dietary soy consumption has deleterious effects on GCT development. PMID:25165122

  5. Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

    PubMed

    Zhang, Ping-Wu; Haidet-Phillips, Amanda M; Pham, Jacqueline T; Lee, Youngjin; Huo, Yuqing; Tienari, Pentti J; Maragakis, Nicholas J; Sattler, Rita; Rothstein, Jeffrey D

    2016-01-01

    Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

  6. Cytotoxic effects of novel phytosphingosine derivatives, including N,N-dimethylphytosphingosine and N-monomethylphytosphingosine, in human leukemia cell line HL60.

    PubMed

    Park, Sook Ryun; Cho, Hyo Jin; Moon, Kyung Jin; Chun, Kyung-Hee; Kong, Sun-Young; Yoon, Sung-Soo; Lee, Jong Seok; Park, Seonyang

    2010-01-01

    Novel phytosphingosine derivatives have been developed based on the inhibition of sphingosine kinase, which has been implicated in cell growth and inhibition of ceramide-mediated apoptosis. This study evaluated the cytotoxic effects and underlying mechanisms of action of novel phytosphingosine derivatives, including N-monomethylphytosphingosine (MMPH) and N,N-dimethylphytosphingosine (DMPH) and the pegylated forms MMPH-PEG and DMPH-PEG, in human leukemia HL60 cells. In viability and proliferation assays using WST-1, all four drugs induced suppression of cell growth and viability in a concentration-dependent manner. Among them, DMPH had the highest antileukemic activity and induced apoptosis via caspase-8, caspase-3, and caspase-9 activation. The apoptotic effect was also associated with Fas/FasL upregulation, Bid cleavage, Bcl-2 downregulation, Bax upregulation, mitochondrial membrane depolarization, and cytochrome c release. DMPH decreased the phosphorylation of ERK and inhibited daunorubicin-induced ERK activation. Furthermore, DMPH displayed synergistic cytotoxicity with daunorubicin in a sequence-dependent manner. Our findings indicate that DMPH has potential as an effective cytotoxic agent for leukemia.

  7. Hydroxy Cinnamic Acid Derivatives as Partial PPARγ agonists: In Silico Studies, Synthesis and Biological Characterization Against Chronic Myeloid Leukemia Cell Line (K562).

    PubMed

    Joshi, Hardik; Marulkar, Kavita; Gota, Vikram; Ramaa, C S

    2016-06-06

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that regulates the expression of many genes relevant to carcinogenesis. By analogy to selective estrogen receptor modulator for treatment of cancer, selective or partial PPARγ agonists are considered clinically important for chemotherapy of cancer. A series of p-coumaric (3a-3y) and ferulic acid (4a-4y) derivatives were designed and docked and virtually studied for their molecular properties. Synthesized derivatives were assessed to check their effect on non-transformed hepatocytes and further evaluated for their anti-proliferative potential on K562. Molecules 3c, 3m, 4c and 4m were found to have GI50 value less than 50μM. These molecules were found to block G0/G1 phase of cell cycle in dose dependent manner. Western blot analysis revealed that these molecules inhibit proliferating cell nuclear antigen (PCNA) and cyclin D1 expression. Collectively, these results suggest that these molecules could play a role as a novel therapeutic strategy for chronic myeloid leukemia.

  8. Comparative cytotoxicity of fumonisin B1 in two cell lines derived from normal human bronchial epithelial cells using four distinct bioassay techniques.

    PubMed

    Lewis, C; Smith, J; Anderson, J; Freshney, R

    1999-06-01

    This study focuses on the cytotoxic effects of fumonisin B1 (FB1) on both immortalised and immortalised and subsequently transfected normal human bronchial epithelial (NHBE) cells of human origin using four bioassays. While the MTT, Neutral Red and hexosaminidase colorimetric assays showed little difference between the toxic effects on the two related cell lines, the clonogenic assay, measuring cell survival and proliferation, indicated that FB1 had a more toxic effect on the nontransfected cells. This kind ofin vitro approach using cells which retain many characteristics of normal cell growth and differentiation can go some way to developing evaluation models for food safety in the case of mycotoxin contamination without resorting totally to whole animal testing. Nevertheless, one or two cytotoxicity tests may be inadequate for a complete appraisal of toxic potential: rather, as wide a range of methodologies as feasible should be employed initially before meaningful conclusions may be drawn.

  9. Analysis of the activation state of alpha4beta1 integrin in human B cell lines derived from myeloma, leukemia or lymphoma.

    PubMed

    García-Gila, M; Cabañas, C; García-Pardo, A

    1997-12-01

    Myeloma cells specifically localize in the bone marrow and rarely circulate in blood. To study whether this immobilization could be partially explained by the presence of constitutively activated integrins, particularly alpha4beta1, we used the activation reporter HUTS-21 anti-beta1 mAb. These analyses showed that beta1 integrins on myeloma cells were moderately active and could be upregulated similarly to integrins on lymphoma or leukemia cells. Myeloma cells were also tested for their ability to attach to RGD-containing fibronectin fragments, a property of activated (but not resting) alpha4beta1. Two cell lines adhered to these fragments and this was inhibited by anti-alpha5 but not by anti-alpha4 mAbs. These results show that myeloma cells bear low/moderately active alpha4beta1 and support the notion that multiple interactions contribute to their localization in the bone marrow.

  10. Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

    SciTech Connect

    Rudzok, S.; Schlink, U.; Herbarth, O.; Bauer, M.

    2010-05-01

    The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-like enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.

  11. Properties of Dense Cores Embedded in Musca Derived from Extinction Maps and 13CO, C18O, and NH3 Emission Lines

    NASA Astrophysics Data System (ADS)

    Machaieie, Dinelsa A.; Vilas-Boas, José W.; Wuensche, Carlos A.; Racca, Germán A.; Myers, Philip C.; Hickel, Gabriel R.

    2017-02-01

    Using near-infrared data from the Two Micron All Sky Survey catalog and the Near Infrared Color Excess method, we studied the extinction distribution in five dense cores of Musca, which show visual extinction greater than 10 mag and are potential sites of star formation. We analyzed the stability in four of them, fitting their radial extinction profiles with Bonnor–Ebert isothermal spheres, and explored their properties using the J = 1–0 transition of 13CO and C18O and the J = K = 1 transition of NH3. One core is not well described by the model. The stability parameter of the fitted cores ranges from 4.5 to 5.7 and suggests that all cores are stable, including Mu13, which harbors one young stellar object (YSO), the IRAS 12322-7023 source. However, the analysis of the physical parameters shows that Mu13 tends to have larger A V, n c, and P ext than the remaining starless cores. The other physical parameters do not show any trend. It is possible that those are the main parameters to explore in active star-forming cores. Mu13 also shows the most intense emission of NH3. Its 13CO and C18O lines have double peaks, whose integrated intensity maps suggest that they are due to the superposition of clouds with different radial velocities seen in the line of sight. It is not possible to state whether these clouds are colliding and inducing star formation or are related to a physical process associated with the formation of the YSO.

  12. Two novel cultured cell lines, A3/Kawakami and A4/Fukuda, derived from malignant lymphoma of B(non-T)-cell nature of the gastrointestinal tract.

    PubMed

    Hirose, M; Minato, K; Tobinai, K; Shimoyama, M; Watanabe, S; Abe, T; Deura, K

    1983-02-01

    A3/Kawakami was derived from ascitic lymphoma cells of a 68-year-old female patient with malignant lymphoma (non-Hodgkin's lymphoma, diffuse, large cell type) of the stomach and A4/Fukuda was derived from ascitic lymphoma cells of a 52-year-old female patient with double cancer of the colon (well-differentiated papillary adenocarcinoma and non-Hodgkin's lymphoma, diffuse, large cell type). The fresh ascitic lymphoma cells in the case of A3/Kawakami were surface immunoglobulin-positive, but the cultured A3/Kawakami cells no longer expressed any distinct markers. In the case of A4/Fukuda, the fresh ascitic lymphoma cells and cultured cells did not express any specific surface markers. Only 20% of A4/Fukuda cells were reactive with OKI1. However, a small amount of IgM could be detected in the cell extract and concentrated culture supernate of A4/Fukuda. In addition, A4/Fukuda cells heterotransplanted into anti-thymocyte sera-treated newborn hamster or athymic nude mice with a BALB/c genetic background were found to have weak surface immunoglobulin and distinct cytoplasmic immunoglobulin (gamma, mu). These data suggest that A4/Fukuda cells share the characteristics of the late differentiation stage of B-cell lineage (intermediate between mature B-cells and plasma cells). It was found that monoclonal antibodies, OKT4 and anti-Leu 3a, which are known to react specifically with inducer/helper T-cells, reacted to both A3/Kawakami and A4/Fukuda cells. The karyotypes of both A3/Kawakami and A4/Fukuda cells were very complicated but included some marker chromosomes such as 14q+.

  13. Synthesis, characterization of 1,2,4-triazole Schiff base derived 3d-metal complexes: Induces cytotoxicity in HepG2, MCF-7 cell line, BSA binding fluorescence and DFT study

    NASA Astrophysics Data System (ADS)

    Tyagi, Prateek; Tyagi, Monika; Agrawal, Swati; Chandra, Sulekh; Ojha, Himanshu; Pathak, Mallika

    2017-01-01

    Two novel Schiff base ligands H2L1 and H2L2 have been synthesized by condensation reaction of amine derivative of 1,2,4-triazole moiety with 2-hydroxy-4-methoxybenzaldehyde. Co(II), Ni(II), Cu(II) and Zn(II) of the synthesized Schiff bases were prepared by using a molar ratio of ligand:metal as 1:1. The structure of the Schiff bases and synthesized metal complexes were established by 1H NMR, UV-Vis, IR, Mass spectrometry and molar conductivity. The thermal stability of the complexes was study by TGA. Fluorescence quenching mechanism of metal complexes 1-4 show that Zn(II) and Cu(II) complex binds more strongly to BSA. In DFT studies the geometries of Schiff bases and metal complexes were fully optimized with respect to the energy using the 6-31 + g(d,p) basis set. The spectral data shows that the ligands behaves as binegative tridentate. On the basis of the spectral studies, TGA and DFT data an octahedral geometry has been assigned for Co(II), Ni(II), square planar for Cu(II) and tetrahedral for Zn(II) complexes. The anticancer activity were screened against human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (Hep-G2). Result indicates that metal complexes shows increase cytotoxicity in proliferation to cell lines as compared to free ligand.

  14. Synthesis, characterization of 1,2,4-triazole Schiff base derived 3d-metal complexes: Induces cytotoxicity in HepG2, MCF-7 cell line, BSA binding fluorescence and DFT study.

    PubMed

    Tyagi, Prateek; Tyagi, Monika; Agrawal, Swati; Chandra, Sulekh; Ojha, Himanshu; Pathak, Mallika

    2017-01-15

    Two novel Schiff base ligands H2L(1) and H2L(2) have been synthesized by condensation reaction of amine derivative of 1,2,4-triazole moiety with 2-hydroxy-4-methoxybenzaldehyde. Co(II), Ni(II), Cu(II) and Zn(II) of the synthesized Schiff bases were prepared by using a molar ratio of ligand:metal as 1:1. The structure of the Schiff bases and synthesized metal complexes were established by (1)H NMR, UV-Vis, IR, Mass spectrometry and molar conductivity. The thermal stability of the complexes was study by TGA. Fluorescence quenching mechanism of metal complexes 1-4 show that Zn(II) and Cu(II) complex binds more strongly to BSA. In DFT studies the geometries of Schiff bases and metal complexes were fully optimized with respect to the energy using the 6-31+g(d,p) basis set. The spectral data shows that the ligands behaves as binegative tridentate. On the basis of the spectral studies, TGA and DFT data an octahedral geometry has been assigned for Co(II), Ni(II), square planar for Cu(II) and tetrahedral for Zn(II) complexes. The anticancer activity were screened against human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (Hep-G2). Result indicates that metal complexes shows increase cytotoxicity in proliferation to cell lines as compared to free ligand.

  15. In vitro anticancer activity of Betulinic acid and derivatives thereof on equine melanoma cell lines from grey horses and in vivo safety assessment of the compound NVX-207 in two horses.

    PubMed

    Liebscher, G; Vanchangiri, K; Mueller, Th; Feige, K; Cavalleri, J-M V; Paschke, R

    2016-02-25

    Betulinic acid, a pentacyclic triterpene, and its derivatives are promising compounds for cancer treatment in humans. Melanoma is not only a problem for humans but also for grey horses as they have a high potential of developing melanoma lesions coupled to the mutation causing their phenotype. Current chemotherapeutic treatment carries the risk of adverse health effects for the horse owner or the treating veterinarian by exposure to antineoplastic compounds. Most treatments have low prospects for systemic tumor regression. Thus, a new therapy is needed. In this in vitro study, Betulinic acid and its two derivatives B10 and NVX-207, both with an improved water solubility compared to Betulinic acid, were tested on two equine melanoma cell lines (MelDuWi and MellJess/HoMelZh) and human melanoma (A375) cell line. We could demonstrate that all three compounds especially NVX-207 show high cytotoxicity on both equine melanoma cell lines. The treatment with these compounds lead to externalization of phosphatidylserines on the cell membrane (AnnexinV-staining), DNA-fragmentation (cell cycle analysis) and activation of initiator and effector caspases (Caspase assays). Our results indicate that the apoptosis is induced in the equine melanoma cells by all three compounds. Furthermore, we succeed in encapsulating the most active compound NVX-207 in 2-Hydroxyprolyl-β-cyclodextrine without a loss of its activity. This formulation can be used as a promising antitumor agent for treating grey horse melanoma. In a first tolerability evaluation in vivo the formulation was administered every one week for 19 consecutive weeks and well tolerated in two adult melanoma affected horses.

  16. Liposome armed with herpes virus-derived gH625 peptide to overcome doxorubicin resistance in lung adenocarcinoma cell lines

    PubMed Central

    Falanga, Annarita; Zappavigna, Silvia; Stiuso, Paola; Tirino, Virginia; Desiderio, Vincenzo; Papaccio, Gianpaolo; Galdiero, Massimiliano; Giordano, Antonio; Galdiero, Stefania; Caraglia, Michele

    2016-01-01

    New delivery systems including liposomes have been developed to circumvent drug resistance. To enhance the antitumor efficacy of liposomes encapsulating anti-cancer agents, we used liposomes externally conjugated to the 20 residue peptide gH625. Physicochemical characterization of the liposome system showed a size of 140 nm with uniform distribution and high doxorubicin encapsulation efficiency. We evaluated the effects of increasing concentrations of liposomes encapsulating Doxo (LipoDoxo), liposomes encapsulating Doxo conjugated to gH625 (LipoDoxo-gH625), empty liposomes (Lipo) or free Doxo on growth inhibition of either wild type (A549) or doxorubicin-resistant (A549 Dx) human lung adenocarcinoma. After 72 h, we found that the growth inhibition induced by LipoDoxo-gH625 was higher than that caused by LipoDoxo with an IC50 of 1 and 0.3 μM in A549 and A549 Dx cells, respectively. The data on cell growth inhibition were paralleled by an higher oxidative stress and an increased uptake of Doxo induced by LipoDoxo-gH625 compared to LipoDoxo, above all in A549 Dx cells. Cytometric analysis showed that the antiproliferative effects of each drug treatment were mainly due to the induction of apoptosis. In conclusion, liposomes armed with gH625 are able to overcome doxorubicin resistance in lung adenocarcinoma cell lines. PMID:26554306

  17. D-1 dopaminergic and beta-adrenergic stimulation of adenylate cyclase in a clone derived from the human astrocytoma cell line G-CCM.

    PubMed

    Balmforth, A J; Ball, S G; Freshney, R I; Graham, D I; McNamee, H B; Vaughan, P F

    1986-09-01

    Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.

  18. Liposome armed with herpes virus-derived gH625 peptide to overcome doxorubicin resistance in lung adenocarcinoma cell lines.

    PubMed

    Perillo, Emiliana; Porto, Stefania; Falanga, Annarita; Zappavigna, Silvia; Stiuso, Paola; Tirino, Virginia; Desiderio, Vincenzo; Papaccio, Gianpaolo; Galdiero, Massimiliano; Giordano, Antonio; Galdiero, Stefania; Caraglia, Michele

    2016-01-26

    New delivery systems including liposomes have been developed to circumvent drug resistance. To enhance the antitumor efficacy of liposomes encapsulating anti-cancer agents, we used liposomes externally conjugated to the 20 residue peptide gH625. Physicochemical characterization of the liposome system showed a size of 140 nm with uniform distribution and high doxorubicin encapsulation efficiency. We evaluated the effects of increasing concentrations of liposomes encapsulating Doxo (LipoDoxo), liposomes encapsulating Doxo conjugated to gH625 (LipoDoxo-gH625), empty liposomes (Lipo) or free Doxo on growth inhibition of either wild type (A549) or doxorubicin-resistant (A549 Dx) human lung adenocarcinoma. After 72 h, we found that the growth inhibition induced by LipoDoxo-gH625 was higher than that caused by LipoDoxo with an IC50 of 1 and 0.3 μM in A549 and A549 Dx cells, respectively. The data on cell growth inhibition were paralleled by an higher oxidative stress and an increased uptake of Doxo induced by LipoDoxo-gH625 compared to LipoDoxo, above all in A549 Dx cells. Cytometric analysis showed that the antiproliferative effects of each drug treatment were mainly due to the induction of apoptosis. In conclusion, liposomes armed with gH625 are able to overcome doxorubicin resistance in lung adenocarcinoma cell lines.

  19. Antibodies binding granulocyte-macrophage colony stimulating factor produced by cord blood-derived B cell lines immortalized by Epstein-Barr virus in vitro.

    PubMed

    Revoltella, R P; Laricchia Robbio, L; Liberati, A M; Reato, G; Foa, R; Funaro, A; Vinante, F; Pizzolo, G

    2000-09-15

    We detected natural antibodies (auto-Abs) binding human granulocyte-macrophage colony stimulating factor (GM-CSF) in umbilical cord blood (CB) (23 of 94 samples screened) and peripheral blood of women at the end of pregnancy (6 of 42 samples tested). To demonstrate that Abs detected in CB were produced by the fetus, CB mononuclear cells were infected with Epstein-Barr virus in vitro. Ten cell lines producing constitutively anti-recombinant human GM-CSF (rhGM-CSF) Abs were isolated and characterized. These cells displayed a male karyotype, an early activated B cell phenotype, coexpressed surface IgM and IgD, and secreted only IgM with prevailing lambda clonal restriction. Specific cell surface binding of biotinylated rhGM-CSF and high-level anti-rhGM-CSF IgM Ab production were typical features of early cell cultures. In late cell passages the frequency of more undifferentiated B cells increased. Serum Abs of either maternal or fetal origin or Abs produced in culture did not affect the granulocyte and macrophage colony stimulating activity of rhGM-CSF from bone marrow progenitors in soft agar, suggesting that the Abs produced were nonneutralizing.

  20. Secular trend of the equilibrium-line altitude on the western side of the southern Andes, derived from radiosonde and surface observations

    NASA Astrophysics Data System (ADS)

    Carrasco, Jorge F.; Osorio, Roberto; Casassa, Gino

    The altitude of the 0°C isotherm obtained from radiosonde data of the aerological Chilean stations Antofagasta, Quintero/Santo Domingo, Puerto Montt and Punta Arenas are analyzed, along with surface temperature and precipitation records from nearby stations. The strong effect of the 1976/77 climate shift due to a change in the Pacific Decadal Oscillation is evident in the temperature and precipitation data. The data are used as input for an empirical model which reconstructs annually the equilibrium-line altitude (ELA) for the last 49 years on the western side of the southern Andes. The model takes air temperature, precipitation and altitude as the main parameters, and was first developed by Fox (1993) and applied by Condom and others (2007). From the radiosonde data, a significant positive trend of the 0°C isotherm has occurred in the northern, central and southern regions, indicating an ELA rise due to regional warming. General glacier retreat, ice thinning and negative mass balance observed during the past few decades in virtually all the Chilean Andes concur with the observed ELA reconstruction. In the Punta Arenas radiosonde record there is slight evidence for precipitation increase but no evidence for significant warming in the past few decades. This results in a slight lowering of the ELA according to the model reconstruction, which does not agree with the strong and increased glacier retreat observed in recent decades in Patagonia.

  1. The effects of retinoic acid on reversing the adipocyte differentiation into an osteoblastic tendency in ST2 cells, a murine bone marrow-derived stromal cell line.

    PubMed

    Ding, J; Woo, J T; Nagai, K

    2001-07-01

    Although the mouse bone marrow stromal cell line ST2 has been known to be differentiated into osteoblasts, the differentiation characteristics of the cell into adipocyte and the concerned relationship between its adipogenesis and osteogenesis remains unknown. The adipogenic induction medium which is made up of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine(IBMX), stimulated the expression of n early adipogenic marker PPAR gamma and a late marker GPDH in ST2 cells. The triglyceride accumulation and lipid stain level generated by the induction medium in ST2 cells was inhibited by RA with IC(50) at about 1 nM. The induction medium up-regulated expression of PPARgamma and GPDH was also inhibited by RA whereas RA (30 nM) exterted no effect on the cell growth. Interestingly, treatment of the cells with induction medium in the presense of RA caused a 3- or 10-fold higher in ALP activity respectively as compared to those treated with RA or the induction medium alone. RT-PCR analysis showed that such a synergistic effect of RA and the induction medium paralleled the process of inhibition on adipogenesis. Additional experiments showed that IBMX played a key role in increasing the effect of RA and ALP activity. Our results suggested that the relationship between adipogenesis and osteogenesis in ST2 cells was reciprocally interrelated and the process of adipogenesis could be potentially reversed into an osteoblastogenic tendency. This is the first report demonstrating that RA transforms adipogenic potential into an osteoblastic tendency.

  2. Cystathionine β-synthase-derived hydrogen sulfide regulates lipopolysaccharide-induced apoptosis of the BRL rat hepatic cell line in vitro.

    PubMed

    Yan, Jun; Teng, Feixiang; Chen, Weiwei; Ji, Yinglei; Gu, Zhenyong

    2012-11-01

    Hydrogen sulfide (H(2)S), is a member of the novel family of endogenous gaseous transmitters, termed "gasotransmitters exhibiting diverse physiological activities, and is generated in mammalian tissues mainly by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) in conjunction with cysteine (aspartate) aminotranferase (CAT). The distributions of these enzymes are species- and tissue-specific. The liver, as the main organ that generates H(2)S in vivo, functions in biotransformation and metabolism. However, the liver is vulnerable to damage from internal and external factors, including inflammatory mediators, drugs and poisons. The present study evaluated the endogenous CBS-H(2)S synthesis regulating lipopolysaccharide (LPS)-induced apoptosis of hepatic cells. The rat hepatic cell line, BRL, was incubated with LPS for various time periods to establish a cell-damage model. Incubation with LPS resulted in a significant increase in CBS expression and H(2)S production. It also stimulated apoptosis and decreased the mitochondrial membrane potential. Pretreatment with the CBS inhibitor aminooxyacetic acid (AOAA) or CBS small interfering RNA (siRNA) decreased LPS-enhanced H(2)S production. Notably, apoptosis increased for a short period and then decreased gradually, while the mitochondrial membrane potential demonstrated the opposite trend. These results showed that endogenous CBS-H(2)S synthesis demonstrated early anti-apoptotic activity and subsequent pro-apoptotic activity in LPS-induced apoptosis. These results suggest a new approach for developing novel drugs for this condition.

  3. Treatment factors influencing the use of recombinant platelet-derived growth factor (Regranex) for head and lateral line erosion syndrome in ocean surgeonfish (Acanthurus bahianus).

    PubMed

    Fleming, Gregory J; Corwin, Allison; McCoy, A Jeanene; Stamper, M Andrew

    2008-06-01

    The clinical efficacy of becaplermin (Regranex, Ortho-McNeil Pharmaceutical, Inc., Raritan, New Jersey 08869) on head and lateral line erosion syndrome (HLLES) in ocean surgeonfish (Acanthurus bahianus) was examined in a two-part study to determine suitable application frequencies of the drug and the effects of the environment on outcome of treatments. In the first part of the study, 12 ocean surgeonfish with prior HLLES were equally divided into three treatment groups, varying in application frequency of becaplermin: 1) one application, 2) three applications every 3 wk, and 3) no applications. After 9 wk, it was determined through photograph and computer analysis that fish treated with becaplermin did heal significantly more than the fish that were not treated. No significant difference was found when comparing the two treatment regimes; therefore, a one-time treatment of becaplermin was just as effective as the three-time application, reducing cost of the treatment, staff labor, and stress to the animals. In the second part of the study, the effect of the water environment on HLLES development was documented before and after a one-time treatment of becaplermin. When fish were treated and placed into a system known to cause HLLES, the becaplermin treatment had no effect on reducing the HLLES progression, whereas treated fish that were placed into a system that did not cause HLLES showed a significant decrease in erosion after the treatment. Therefore, treating fish being placed into a HLLES-causing system serves no beneficial purpose to healing HLLES.

  4. Fern plant-derived protoapigenone leads to DNA damage, apoptosis, and G(2)/m arrest in lung cancer cell line H1299.

    PubMed

    Chiu, Chien-Chih; Chang, Hsueh-Wei; Chuang, Da-Wei; Chang, Fang-Rong; Chang, Yu-Ching; Cheng, Yu-Shan; Tsai, Ming-Tz; Chen, Wan-Yu; Lee, Su-Shuo; Wang, Chih-Kuang; Chen, Jeff Yi-Fu; Wang, Hui-Min; Chen, Chao-Chieh; Liu, Yin-Chang; Wu, Yang-Chang

    2009-10-01

    Protoapigenone, isolated from the native fern plant Thelypteris torresiana, has anticancer activity against some cancer cells. However, the toxicological mechanism for protoapigenone is still unknown. Here, we investigated the anticancer effect of protoapigenone on human lung cancer cell lines. The comet assay showed that DNA damage induced by protoapigenone is dose-dependent. Trypan blue exclusion showed that the cell killing by protoapigenone is both time and dose dependent. The IC(50) of protoapigenone for 12, 24, and 48 h in H1299 cells is 6.11, 2.74, and 1.49 microM, respectively. Flow cytometry showed cell cycle perturbation such as sub-G(1) accumulation (at 1.57 microM for 48 h and at 3.57 microM for 12 and 24 h) and G(2)/M arrest (at 3.57 microM for 12 and 24 h) for protoapigenone. The sub-G(1) accumulation phenomena in the 3.57 microM for 24 h sample were shown to be apoptosis using Annexin V-immunofluorescence/propidium iodide staining. These results suggest protoapigenone is a potential chemotherapeutic agent for lung cancers.

  5. Characterization of arsenic trioxide resistant clones derived from Jurkat leukemia T cell line: focus on PI3K/Akt signaling pathway.

    PubMed

    Roszak, Joanna; Smok-Pieniążek, Anna; Nocuń, Marek; Stępnik, Maciej

    2013-10-05

    In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8-12weeks in the presence of 1, 2.5 and 5μM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a

  6. Human omental adipose-derived mesenchymal stem cell-conditioned medium alters the proteomic profile of epithelial ovarian cancer cell lines in vitro

    PubMed Central

    Zhang, Yanling; Dong, Weihong; Wang, Junjie; Cai, Jing; Wang, Zehua

    2017-01-01

    Mesenchymal stem cells (MSCs) have been reported to participate in the formation of supportive tumor stroma. The abilities of proliferation and invasion of human epithelial ovarian cancer (EOC) cells were significantly enhanced when indirectly cocultured with human omental adipose-derived MSCs (O-ADSCs) in vitro. However, the underlying mechanisms remain poorly understood. In this study, EOC cells were cultured with conditioned medium (CM) from O-ADSCs (O-ADSC), and the effect of O-ADSC CM on the proteomic profile of EOC cells was assessed by two-dimensional gel electrophoresis (2-DE), followed by liquid chromatography and tandem mass spectrometry. The 2-DE assays revealed a global increase in protein expression in the EOC cells treated with CM. Nine proteins were identified from 11 selected protein spots with differential expression after treatment with CM from O-ADSCs. All the nine proteins have been linked to carcinoma and apoptosis, and the migration abilit