Science.gov

Sample records for rodent cell lines

  1. Absence of keratins 8 and 18 in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: Cell line selective effects on cell invasion.

    PubMed

    Kwan, Raymond; Looi, Kok Sun; Omary, M Bishr

    2015-07-01

    Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 mRNA levels being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription and mRNA or protein stability. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Absence of keratins 8 and 18 expression in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: cell line selective effects on cell invasion

    PubMed Central

    Omary, M. Bishr

    2016-01-01

    Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription or mRNA stability. PMID:25882495

  3. Digoxin reduces the mutagenic effects of Mitomycin C in human and rodent cell lines.

    PubMed

    de Oliveira, Júlia Teixeira; Barbosa, Maria C da Silva; de Camargos, Luiz F; da Silva, Isabella Viana Gomes; Varotti, Fernando de Pilla; da Silva, Luciana M; Moreira, Leonardo Marmo; Lyon, Juliana Pereira; Dos Santos, Vanessa J da Silva Vieira; Dos Santos, Fabio Vieira

    2017-03-20

    Digoxin is a drug widely used to treat heart failure and studies have demonstrated its potential as anticancer agent. In addition, digoxin presents the potential to interact with a series of other compounds used in medicine. The aim of the present study was to evaluate in vitro the cytotoxicity, genotoxicity and mutagenicity of digoxin and its potential to interact with the mutagen Mitomycin C (MMC). The cytotoxicity of digoxin was assessed by employing the MTT method and the comet assay was performed to assess the genotoxicity of this medicine in CHO-K1 and HeLa cell lines. Besides, the cytokinesis-block micronucleus assay was performed to assess the mutagenicity and the antimutagenicity of this drug. The Ames assay was also performed with TA98 and TA100 strains of S. typhimurium. Results showed that digoxin was cytotoxic, genotoxic and mutagenic for HeLa and CHO-K1 cell lines at concentrations many times higher than those observed in human therapeutic conditions. Nevertheless, an antimutagenic effect against the mutagen MMC was observed on both cell lines in concentrations near those used therapeutically in humans. This chemoprotective effect observed is an interesting finding that should be better explored regarding its impact in anticancer chemotherapy.

  4. 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.

  5. Saffold virus is able to productively infect primate and rodent cell lines and induces apoptosis in these cells

    PubMed Central

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Tan, Yee Joo; Chua, Kaw Bing

    2014-01-01

    Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage. PMID:26038510

  6. Saffold virus is able to productively infect primate and rodent cell lines and induces apoptosis in these cells.

    PubMed

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Tan, Yee Joo; Chua, Kaw Bing

    2014-02-01

    Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage.

  7. Analysis of DNA strand breaks induced in rodent liver in vivo, hepatocytes in primary culture, and a human cell line by chlorinated acetic acids and chlorinated acetaldehydes

    SciTech Connect

    Chang, L.W.; Daniel, F.B. ); DeAngelo, A.B. )

    1992-01-01

    An alkaline unwinding assay was used to quantitate the induction of DNA strand breaks (DNA SB) in the livers of rats and mice treated in vivo, in rodent hepatocytes in primary culture, and in CCRF-CEM cells, a human lymphoblastic leukemia cell line, following treatment with tri-(TCA), di-(CA), and mono-(MCA) chloroacetic acid and their corresponding aldehydes, tri-(chloralhydrate, CH), di(DCAA) and mono-(CAA) chloroacetaldehyde. None of the chloracetic acids induced DNA SB in the livers of rats at 4 hr following a single administration of 1-10 mmole/kg. TCA (10 mmole/kg) and DCA (5 and 10 mmole/kg) did produce a small amount of strand breakage in mice (7% at 4hr) but not at 1 hr. N-nitrosodiethylamine (DENA), an established alkylating agent and a rodent hepatocarcinogen, produced DNA SB in the livers of both species. TCA, DCA, and MCA also failed to induce DNA strand breaks in splenocytes and epithelial cells derived from the stomach and duodenum of mice treated in vivo. None of the three chloroacetaldehydes induced DNA SB in either mouse or rat liver. These studies provide further evidence that the chloroacetic acids lack genotoxic activity not only in rodent liver, a tissue in that they induce tumors, but in a variety of other rodent tissues and cultured cell types. Two of the chloroacetaldehydes, DCAA and CAA, are direct acting DNA damaging agents in CCRF-CEM cells, but not in liver or splenocytes in vivo or in cultured hepatocytes. CH showed no activity in any system investigated. 58 refs., 6 figs., 2 tabs.

  8. Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line.

    PubMed

    Schilling, Birgit; Bharath M M, Srinivas; Row, Richard H; Murray, James; Cusack, Michael P; Capaldi, Roderick A; Freed, Curt R; Prasad, Kedar N; Andersen, Julie K; Gibson, Bradford W

    2005-01-01

    Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

  9. The effect of 50 Hz electromagnetic fields on the formation of micronuclei in rodent cell lines exposed to gamma radiation.

    PubMed

    Lagroye, I; Poncy, J L

    1997-08-01

    Low frequency electromagnetic fields (EMF) do not produce enough energy to damage DNA, in contrast to ionizing radiations. Any relationship between increased incidence of cancer and EMF must therefore be explained by a promoting effect on cellular transformation by ionizing radiation. The aim of this study was to investigate using the cytokinesis-blocked micronucleus assay a possible amplification of the genotoxic effects of ionizing radiations in cells exposed to combined static and power-frequency electromagnetic fields. Rat tracheal epithelial cell lines were first exposed in vitro to 60Co gamma rays (0, 2 and 6 Gy) and cells were then cultured for 24 h in a homogeneous sinusoidal 50 Hz magnetic field (flux density: 100 microTrms) combined with an artificial geomagnetic-like field created by the use of horizontal and vertical pairs of Helmholtz coils. Control cells were cultured in an adjacent incubator where the background EMF was about 0.1 microTrms. Under our in vitro experimental conditions, EMF appeared to have no significant direct effect on micronucleus induction in rat tracheal cell lines. However, an increased frequency of binucleated cells with micronuclei was observed in cells exposed to 6 Gy of gamma rays and EMF, compared with gamma irradiation alone. This could enhance radiation-induced genomic alterations and increase the probability of neoplastic transformation.

  10. The response of human and rodent cells to hyperthermia

    SciTech Connect

    Roizin-Towle, L.; Pirro, J.P. )

    1991-04-01

    Inherent cellular radiosensitivity in vitro has been shown to be a good predictor of human tumor response in vivo. In contrast, the importance of the intrinsic thermosensitivity of normal and neoplastic human cells as a factor in the responsiveness of human tumors to adjuvant hyperthermia has never been analyzed systematically. A comparison of thermal sensitivity and thermo-radiosensitization in four rodent and eight human-derived cell lines was made in vitro. Arrhenius plots indicated that the rodent cells were more sensitive to heat killing than the human, and the break-point was 0.5 degrees C higher for the human than rodent cells. The relationship between thermal sensitivity and the interaction of heat with X rays at low doses was documented by thermal enhancement ratios (TER's). Cells received either a 1 hr exposure to 43 degrees C or a 20 minute treatment at 45 degrees C before exposure to 300 kVp X rays. Thermal enhancement ratios ranged from 1.0 to 2.7 for human cells heated at 43 degrees C and from 2.1 to 5.3 for heat exposures at 45 degrees C. Thermal enhancement ratios for rodent cells were generally 2 to 3 times higher than for human cells, because of the fact that the greater thermosensitivity of rodent cells results in a greater enhancement of radiation damage. Intrinsic thermosensitivity of human cells has relevance to the concept of thermal dose; intrinsic thermo-radiosensitization of a range of different tumor cells is useful in documenting the interactive effects of radiation combined with heat.

  11. Use of resveratrol to improve the effectiveness of cisplatin and doxorubicin: study in human gynecologic cancer cell lines and in rodent heart.

    PubMed

    Rezk, Youssef A; Balulad, Sujata S; Keller, Rebecca S; Bennett, James A

    2006-05-01

    The purpose of this study was to investigate whether resveratrol adds to the growth inhibitory effects of cisplatin and doxorubicin on ovarian and uterine cancer cells and to evaluate whether resveratrol diminishes the cardiac toxicity of doxorubicin in rodent heart. Human ovarian (OVCAR-3) and uterine (Ishikawa) cancer cells in culture were treated with cisplatin and doxorubicin, respectively, with and without resveratrol; and cell growth and viability were evaluated. Neonatal rat ventricular myocytes received doxorubicin in the presence and absence of resveratrol, and cell viability was evaluated. Mice received doxorubicin +/- resveratrol, and electrocardiograms were evaluated. Data were analyzed with analysis of variance and Scheffe's test. Resveratrol combined with cisplatin or with doxorubicin demonstrated an additive growth-inhibitory anticancer effect with a left shift of the cisplatin and doxorubicin dose/response curves. Resveratrol increased the viability of neonatal rat ventricular myocytes that were treated with doxorubicin and reduced doxorubicin-induced bradycardia and QTc interval prolongation in mice. Resveratrol adds to the growth inhibitory/anticancer activity of cisplatin and doxorubicin in vitro and protects against doxorubicin-induced cardiac toxicity both in vitro and in mice.

  12. Isolating human DNA repair genes using rodent-cell mutants

    SciTech Connect

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  13. Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

    PubMed Central

    MacAuley, Alasdair; Cross, James C.; Werb, Zena

    1998-01-01

    Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle. PMID:9529378

  14. Goniothalamin prevents the development of chemically induced and spontaneous colitis in rodents and induces apoptosis in the HT-29 human colon tumor cell line.

    PubMed

    Vendramini-Costa, Débora Barbosa; Alcaide, Antonio; Pelizzaro-Rocha, Karin Juliane; Talero, Elena; Ávila-Román, Javier; Garcia-Mauriño, Sofia; Pilli, Ronaldo Aloise; de Carvalho, João Ernesto; Motilva, Virginia

    2016-06-01

    Colon cancer is the third most incident type of cancer worldwide. One of the most important risk factors for colon cancer development are inflammatory bowel diseases (IBD), thus therapies focusing on IBD treatment have great potential to be used in cancer prevention. Nature has been a source of new therapeutic and preventive agents and the racemic form of the styryl-lactone goniothalamin (GTN) has been shown to be a promising antiproliferative agent, with gastroprotective, antinociceptive and anti-inflammatory effects. As inflammation is a well-known tumor promoter, the major goal of this study was to evaluate the therapeutic and preventive potentials of GTN on chemically induced and spontaneous colitis, as well as the cytotoxic effects of GTN on a human colon tumor cell line (HT-29). GTN treatments inhibited TNBS-induced acute and chronic colitis development in Wistar rats, reducing myeloperoxidase levels and inflammatory cells infiltration in the mucosa. In spontaneous-colitis using IL-10 deficient mice (C57BL/6 background), GTN prevented colitis development through downregulation of TNF-α, upregulation of SIRT-1 and inhibition of proliferation (PCNA index), without signs of toxicity after three months of treatment. In HT-29 cells, treatment with 10μM of GTN induced apoptosis by increasing BAX/BCL2, p-JNK1/JNK1, p-P38/P38 ratios as well as through ROS generation. Caspase 8, 9 and 3 activation also occurred, suggesting caspase-dependent apoptotic pathway, culminating in PARP-1 cleavage. Together with previous data, these results show the importance of GTN as a pro-apoptotic, preventive and therapeutic agent for IBD and highlight its potential as a chemopreventive agent for colon cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. 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.

  16. Identification of aromatase activity in rodent pituitary cell strains.

    PubMed

    Callard, G V; Petro, Z; Tashjian, A H

    1983-07-01

    To date, biochemical evidence has been presented for hypophysial aromatization in only one species, a teleost fish, although the pituitary glands of several mammals have been reported to be aromatase negative. To reinvestigate this problem, established clonal strains of rodent pituitary cells (GH3, GH4C1, and AtT20/D16) were incubated at 37 C for 6-48 h in serum-less medium containing [7-3H]androstenedione. Radiolabeled metabolites were isolated by solvent extraction, thin layer chromatography, and phenolic partition. The authenticity of the estrogenic products in both cells and incubation medium was verified by methylation and recrystallization to constant specific activity. Measurement of androgen metabolites was also validated by recrystallization of selected samples. Authentic estrone and 17 beta-estradiol were identified in cultures of the two PRL- and GH-secreting clones, and there were strain differences in the quantity of estrogen produced (GH3 greater than GH4C1). Under the same conditions, aromatization was not detectable in the ACTH-secreting line (AtT20/D16). A time-yield analysis of androgen metabolism in GH4C1 cells showed that aromatization was linear for 12 h after labeling, but that substrate was diverted mainly to 5 alpha-reducing pathways. Large amounts of highly polar metabolites accumulated 24 and 48 h after the addition of [3H]androgen, and subsequent hydrolysis revealed that these were sulfo- and glucuronoconjugates. The metabolic fate of estrogen in GH4C1 cultures was investigated indirectly by adding a radioinert estrone trap together with the radiolabeled androgen substrate and was also tested in separate cultures by adding [3H]estrone and [3H]estradiol directly. Although the two estrogens were interconverted, there was no evidence that formed or added estrogen was extensively metabolized or conjugated. We conclude that the expression of aromatase activity in hypophysial cells is not a property of all transformed lines but may be dictated

  17. Probabilistic Learning by Rodent Grid Cells

    PubMed Central

    Cheung, Allen

    2016-01-01

    Mounting evidence shows mammalian brains are probabilistic computers, but the specific cells involved remain elusive. Parallel research suggests that grid cells of the mammalian hippocampal formation are fundamental to spatial cognition but their diverse response properties still defy explanation. No plausible model exists which explains stable grids in darkness for twenty minutes or longer, despite being one of the first results ever published on grid cells. Similarly, no current explanation can tie together grid fragmentation and grid rescaling, which show very different forms of flexibility in grid responses when the environment is varied. Other properties such as attractor dynamics and grid anisotropy seem to be at odds with one another unless additional properties are assumed such as a varying velocity gain. Modelling efforts have largely ignored the breadth of response patterns, while also failing to account for the disastrous effects of sensory noise during spatial learning and recall, especially in darkness. Here, published electrophysiological evidence from a range of experiments are reinterpreted using a novel probabilistic learning model, which shows that grid cell responses are accurately predicted by a probabilistic learning process. Diverse response properties of probabilistic grid cells are statistically indistinguishable from rat grid cells across key manipulations. A simple coherent set of probabilistic computations explains stable grid fields in darkness, partial grid rescaling in resized arenas, low-dimensional attractor grid cell dynamics, and grid fragmentation in hairpin mazes. The same computations also reconcile oscillatory dynamics at the single cell level with attractor dynamics at the cell ensemble level. Additionally, a clear functional role for boundary cells is proposed for spatial learning. These findings provide a parsimonious and unified explanation of grid cell function, and implicate grid cells as an accessible neuronal population

  18. Probabilistic Learning by Rodent Grid Cells.

    PubMed

    Cheung, Allen

    2016-10-01

    Mounting evidence shows mammalian brains are probabilistic computers, but the specific cells involved remain elusive. Parallel research suggests that grid cells of the mammalian hippocampal formation are fundamental to spatial cognition but their diverse response properties still defy explanation. No plausible model exists which explains stable grids in darkness for twenty minutes or longer, despite being one of the first results ever published on grid cells. Similarly, no current explanation can tie together grid fragmentation and grid rescaling, which show very different forms of flexibility in grid responses when the environment is varied. Other properties such as attractor dynamics and grid anisotropy seem to be at odds with one another unless additional properties are assumed such as a varying velocity gain. Modelling efforts have largely ignored the breadth of response patterns, while also failing to account for the disastrous effects of sensory noise during spatial learning and recall, especially in darkness. Here, published electrophysiological evidence from a range of experiments are reinterpreted using a novel probabilistic learning model, which shows that grid cell responses are accurately predicted by a probabilistic learning process. Diverse response properties of probabilistic grid cells are statistically indistinguishable from rat grid cells across key manipulations. A simple coherent set of probabilistic computations explains stable grid fields in darkness, partial grid rescaling in resized arenas, low-dimensional attractor grid cell dynamics, and grid fragmentation in hairpin mazes. The same computations also reconcile oscillatory dynamics at the single cell level with attractor dynamics at the cell ensemble level. Additionally, a clear functional role for boundary cells is proposed for spatial learning. These findings provide a parsimonious and unified explanation of grid cell function, and implicate grid cells as an accessible neuronal population

  19. Cell line provenance.

    PubMed

    Freshney, R Ian

    2002-07-01

    Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology. However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere in this volume, the technology, both simple and sophisticated, is available to remedy the problems of misidentification and contamination, given the will to apply it. Combined with proper records of the origin and history of the cell line, assays for authentication and contamination contribute to the provenance of the cell line. Detailed records should start from the initiation or receipt of the cell line, and should incorporate data on the donor as well as the tissue from which the cell line was derived, should continue with details of maintenance, and include any accidental as well as deliberate deviations from normal maintenance. Records should also contain details of authentication and regular checks for contamination. With this information, preferably stored in a database, and suitable backed up, the provenance of the cell line so created makes the cell line a much more valuable resource, fit for validation in industrial applications and more likely to provide reproducible experimental results when disseminated for research in other laboratories.

  20. Cytotoxic and aryl hydrocarbon hydroxylase-inducing effects of laboratory rodent diets. A cell culture study

    SciTech Connect

    Toerroenen, R.; Pelkonen, K.; Kaerenlampi, S. )

    1991-01-01

    Extracts of several rodent diets were studied for their cytotoxic and aryl hydrocarbon hydroxylase-inducing properties by an in vitro method. The cell culture system based on a mouse hepatoma cell line (Hepa-1) was shown to be convenient and sensitive method for screening of diets for these parameters implying the presence of compounds potentially harmful in vivo. Considerable differences among diets and batches were detected. Smallest effects were observed with a semipurified diet and with the unrefined diet which - contrary to other four unrefined diets - contained no fish.

  1. Purification of endothelial cells from rodent brain by immunopanning.

    PubMed

    Zhou, Lu; Sohet, Fabien; Daneman, Richard

    2014-01-01

    This protocol describes the use of immunopanning to purify endothelial cells from the rodent brain. Immunopanning permits the prospective isolation of endothelial cells from nervous tissue by relying on the binding of the endothelial cells to an anti-CD31 antibody adhered to a Petri dish. The cells are viable at the end of this gentle procedure, and they can be analyzed acutely for gene expression or cultured alone or in coculture with other central nervous system (CNS) cell types, including CNS pericytes and CNS astrocytes. This procedure can be used to isolate endothelial cells from either rat or mouse. We have suggested specific antibodies that work for each species. Note that endothelial cells from rats and mice have different morphologies; in general, rat CNS endothelial cells are longer and thinner than mouse CNS endothelial cells. This procedure can also be used to purify endothelial cells from different regions of the CNS, including brain and optic nerve. Dissociation procedures must be optimized for each tissue.

  2. Mechanisms and chemical induction of aneuploidy in rodent germ cells

    SciTech Connect

    Mailhes, J B; Marchetti, F

    2004-10-15

    The objective of this review is to suggest that the advances being made in our understanding of the molecular events surrounding chromosome segregation in non-mammalian and somatic cell models be considered when designing experiments for studying aneuploidy in mammalian germ cells. Accurate chromosome segregation requires the temporal control and unique interactions among a vast array of proteins and cellular organelles. Abnormal function and temporal disarray among these, and others to be inidentified, biochemical reactions and cellular organelles have the potential for predisposing cells to aneuploidy. Although numerous studies have demonstrated that certain chemicals (mainly those that alter microtubule function) can induce aneuploidy in mammalian germ cells, it seems relevant to point out that such data can be influenced by gender, meiotic stage, and time of cell-fixation post-treatment. Additionally, a consensus has not been reached regarding which of several germ cell aneuploidy assays most accurately reflects the human condition. More recent studies have shown that certain kinase, phosphatase, proteasome, and topoisomerase inhibitors can also induce aneuploidy in rodent germ cells. We suggest that molecular approaches be prudently incorporated into mammalian germ cell aneuploidy research in order to eventually understand the causes and mechanisms of human aneuploidy. Such an enormous undertaking would benefit from collaboration among scientists representing several disciplines.

  3. Transgenic rodent assay for quantifying male germ cell mutant frequency.

    PubMed

    O'Brien, Jason M; Beal, Marc A; Gingerich, John D; Soper, Lynda; Douglas, George R; Yauk, Carole L; Marchetti, Francesco

    2014-08-06

    De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.

  4. Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

    PubMed Central

    O'Brien, Jason M.; Beal, Marc A.; Gingerich, John D.; Soper, Lynda; Douglas, George R.; Yauk, Carole L.; Marchetti, Francesco

    2014-01-01

    De novo mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro positive selection assay to measure in vivo mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources. PMID:25145276

  5. CLO: The cell line ontology

    PubMed Central

    2014-01-01

    Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

  6. Stem cell derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model

    PubMed Central

    Donegan, Jennifer J.; Tyson, Jennifer A.; Branch, Sarah Y.; Beckstead, Michael J.; Anderson, Stewart A.; Lodge, Daniel J.

    2016-01-01

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol (MAM) rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity, and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia. PMID:27480492

  7. Comparison of intracerebral transplantation effects of different stem cells on rodent stroke models.

    PubMed

    Wu, Yun; Wu, Jianyu; Ju, Rongkai; Chen, Zhiguo; Xu, Qunyuan

    2015-06-01

    In the present study, induced pluripotent stem cells (iPSCs), induced neural stem cells (iNSCs), mesenchymal stem cells (MSCs) and an immortalized cell line (RMNE6), representing different characteristics of stem cells, were transplanted into normal and/or injured brain areas of rodent stroke models, and their effects were compared to select suitable stem cells for cell replacement stroke therapy. The rat and mice ischaemic models were constructed using the middle cerebral artery occlusion technique. Both electrocoagulation of the artery and the intraluminal filament technique were used. The behaviour changes and fates of grafted stem cells were determined mainly by behaviour testing and immunocytochemistry. Following iPSC transplantation into the corpora striata of normal mice, a tumour developed in the brain. The iNSCs survived well and migrated towards the injured area without differentiation. Although there was no tumourigenesis in the brain of normal or ischaemic mice after the iNSCs were transplanted in the cortices, the behaviour in ischaemic mice was not improved. Upon transplanting MSC and RMNE6 cells into ischaemic rat brains, results similar to iNSCs in mice were seen. However, transplantation of RMNE6 caused a brain tumour. Thus, tumourigenesis and indeterminate improvement of behaviour are challenging problems encountered in stem cell therapy for stroke, and the intrinsic characteristics of stem cells should be remodelled before transplantation. © 2015 The Authors Cell Biochemistry and Function Published by John Wiley & Sons Ltd.

  8. Effect of radiofrequency radiation on MRNA expression in cultured rodent cells

    SciTech Connect

    Parker, J.E.; Kiel, J.L.; Winters, W.D.

    1988-01-01

    Radiofrequency radiation (RFR) has been reported to induce adverse effects in biological systems, such as teratogenic and embryo lethal effects in mammals particularly during exposures producing significant hyperthermia. Other studies have implicated microwave exposure with causing changes in chromosome number and structure, formation of cataracts in humans rabbits and dogs; and promoting malignant tumor formation in rats, as well as increasing tumor production and leukemias. In addition, microwave exposures have been reported to change the structure of purified double-stranded plasmid DNA, causing it to become nicked and increasing the proportion of relaxed to super coiled molecules. In view of these reports of changes at different levels of cellular function and structure of mammalian systems to microwaves, the authors asked themselves if changes at the level of mRNA expression could be detected after microwave exposure of cultured rodent cells. They chose to look at the mRNA expression of certain oncogenes known to show elevated levels during cell replication, at the heat shock proteins known to respond to stresses other than heat, and at the long terminal repeat (LTR) region of mouse mammary tumor virus in four rodent cell lines.

  9. Genotoxic Changes to Rodent Cells Exposed in Vitro to Tungsten, Nickel, Cobalt and Iron

    PubMed Central

    Bardack, Stephanie; Dalgard, Clifton L.; Kalinich, John F.; Kasper, Christine E.

    2014-01-01

    Tungsten-based materials have been proposed as replacements for depleted uranium in armor-penetrating munitions and for lead in small-arms ammunition. A recent report demonstrated that a military-grade composition of tungsten, nickel, and cobalt induced a highly-aggressive, metastatic rhabdomyosarcoma when implanted into the leg muscle of laboratory rats to simulate a shrapnel wound. The early genetic changes occurring in response to embedded metal fragments are not known. In this study, we utilized two cultured rodent myoblast cell lines, exposed to soluble tungsten alloys and the individual metals comprising the alloys, to study the genotoxic effects. By profiling cell transcriptomes using microarray, we found slight, yet distinct and unique, gene expression changes in rat myoblast cells after 24 h metal exposure, and several genes were identified that correlate with impending adverse consequences of ongoing exposure to weapons-grade tungsten alloy. These changes were not as apparent in the mouse myoblast cell line. This indicates a potential species difference in the cellular response to tungsten alloy, a hypothesis supported by current findings with in vivo model systems. Studies examining genotoxic-associated gene expression changes in cells from longer exposure times are warranted. PMID:24619124

  10. Genotoxic changes to rodent cells exposed in vitro to tungsten, nickel, cobalt and iron.

    PubMed

    Bardack, Stephanie; Dalgard, Clifton L; Kalinich, John F; Kasper, Christine E

    2014-03-10

    Tungsten-based materials have been proposed as replacements for depleted uranium in armor-penetrating munitions and for lead in small-arms ammunition. A recent report demonstrated that a military-grade composition of tungsten, nickel, and cobalt induced a highly-aggressive, metastatic rhabdomyosarcoma when implanted into the leg muscle of laboratory rats to simulate a shrapnel wound. The early genetic changes occurring in response to embedded metal fragments are not known. In this study, we utilized two cultured rodent myoblast cell lines, exposed to soluble tungsten alloys and the individual metals comprising the alloys, to study the genotoxic effects. By profiling cell transcriptomes using microarray, we found slight, yet distinct and unique, gene expression changes in rat myoblast cells after 24 h metal exposure, and several genes were identified that correlate with impending adverse consequences of ongoing exposure to weapons-grade tungsten alloy. These changes were not as apparent in the mouse myoblast cell line. This indicates a potential species difference in the cellular response to tungsten alloy, a hypothesis supported by current findings with in vivo model systems. Studies examining genotoxic-associated gene expression changes in cells from longer exposure times are warranted.

  11. Pediatric brain tumor cell lines.

    PubMed

    Xu, Jingying; Margol, Ashley; Asgharzadeh, Shahab; Erdreich-Epstein, Anat

    2015-02-01

    Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research.

  12. RODENT LEYDIG CELL TUMORIGENESIS: A REVIEW OF THE PHYSIOLOGY, PATHOLOGY, MECHANISMS, AND RELEVANCE TO HUMANS

    EPA Science Inventory

    Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has increased in recent years bec...

  13. RODENT LEYDIG CELL TUMORIGENESIS: A REVIEW OF THE PHYSIOLOGY, PATHOLOGY, MECHANISMS, AND RELEVANCE TO HUMANS

    EPA Science Inventory

    Leydig cells (LCs) are the cells of the testis that have as their primary function the production of testosterone. LCs are a common target of compounds tested in rodent carcinogenicity bioassays. The number of reviews on Leydig cell tumors (LCTs) has increased in recent years bec...

  14. Studies on microperoxisomes. VII. Pigment epithelial cells and other cell types in the retina of rodents

    PubMed Central

    1975-01-01

    The pigment epithelial cell of the retina actively participates in two aspects of lipid metabolism: (a) the fatty acid esterification of vitamin A and its storage and transport to the photoreceptors, and (b) the phagocytosis and degradation of the lipoprotein membrane disks shed from the photoreceptor cells. Study of the pigment epithelial cells of adult albino and pigmented rodents has revealed the abundance of an organelle, microperoxisomes, not previously known to exist in this cell type. The metabolism, transport, and storage of lipids are major functions of other cell types which possess large numbers of microperoxisomes associated with a highly developed smooth endoplasmic reticulum. Microperoxisomes were encountered, but relatively rarely, in Muller cells and vascular endothelial cells. A tubular system in photoreceptor terminals is reactive in the cytochemical procedure used to visualize microperoxisomes. PMID:1168648

  15. Cellular Moloney murine sarcoma (c-mos) sequences are hypermethylated and transcriptionally silent in normal and transformed rodent cells

    SciTech Connect

    Gattoni, S.; Kirschmeier, P.; Weinstein, I.B.; Escobedo, J.; Dina, D.

    1982-01-01

    Moloney murine sarcoma virus carries an oncogenic sequence (v-mos) which is homologous to a single copy gene (c-mos) present in the normal cells of several vertebrate species. Because of the possible significance of c-mos sequences in normal development and malignant transformation induced by physical or chemical agents, the authors examined the state of integration, methylation, and transcriptional activity of c-mos sequences in a variety of normal rodent tissues, normal cell lines, or cell lines transformed by radiation or chemical carcinogens. DNA-DNA hybridization, utilizing the Southern blotting technique and a plasmid-derived DNA probe representing the v-mos sequence, gave no evidence for rearrangements of the c-mos sequence in the DNAs obtained from these diverse cell types. Parallel studies employing the restriction enzyme isoschizomers HpaII and MspI indicated that in all of these cell types the c-mos sequences were heavily methylated. In addition, analysis of cellular RNAs by blot hybridization with the v-mos probe failed to detect evidence of transcription of the c-mos sequences in any of these cell types. This was in contrast to a Moloney sarcoma virus-transformed cell line in which they found that the integrated v-mos sequence was both undermethylated and extensively transcribed. Thus, it would appear that c-mos sequences do not play a role in the transformation of rodent cells by chemical or physical agents, although the possible role of other endogenous onc sequences remains to be determined.

  16. Dendritic Arborization Patterns of Small Juxtaglomerular Cell Subtypes within the Rodent Olfactory Bulb

    PubMed Central

    Bywalez, Wolfgang G.; Ona-Jodar, Tiffany; Lukas, Michael; Ninkovic, Jovica; Egger, Veronica

    2017-01-01

    Within the glomerular layer of the rodent olfactory bulb, numerous subtypes of local interneurons contribute to early processing of incoming sensory information. Here we have investigated dopaminergic and other small local juxtaglomerular cells in rats and mice and characterized their dendritic arborization pattern with respect to individual glomeruli by fluorescent labeling via patching and reconstruction of dendrites and glomerular contours from two-photon imaging data. Dopaminergic neurons were identified in a transgenic mouse line where the expression of dopamine transporter (DAT) was labeled with GFP. Among the DAT+ cells we found a small short-axon cell (SAC) subtype featuring hitherto undescribed dendritic specializations. These densely ramifying structures clasped mostly around somata of other juxtaglomerular neurons, which were also small, non-dopaminergic and to a large extent non-GABAergic. Clasping SACs were observed also in wild-type mice and juvenile rats. In DAT+ SAC dendrites, single backpropagating action potentials evoked robust calcium entry throughout both clasping and non-clasping compartments. Besides clasping SACs, most other small neurons either corresponded to the classical periglomerular cell type (PGCs), which was never DAT+, or were undersized cells with a small dendritic tree and low excitability. Aside from the presence of clasps in SAC dendrites, many descriptors of dendritic morphology such as the number of dendrites and the extent of branching were not significantly different between clasping SACs and PGCs. However, a detailed morphometric analysis in relation to glomerular contours revealed that the dendrites of clasping SACs arborized mostly in the juxtaglomerular space and never entered more than one glomerulus (if at all), whereas most PGC dendrites were restricted to their parent glomerulus, similar to the apical tufts of mitral cells. These complementary arborization patterns might underlie a highly complementary functional

  17. CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells

    PubMed Central

    Poeschla, Eric M.; Looney, David J.

    1998-01-01

    A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors. PMID:9658135

  18. Lipopolysaccharide stimulates adrenal steroidogenesis in rodent cells by a NFκB-dependent mechanism involving COX-2 activation.

    PubMed

    Martinez Calejman, C; Astort, F; Di Gruccio, J M; Repetto, E M; Mercau, M; Giordanino, E; Sanchez, R; Pignataro, O; Arias, P; Cymeryng, C B

    2011-04-30

    Stimulation of adrenal steroidogenesis is involved in the HPA response to exogenous noxa. Although inflammatory cytokines can mediate the LPS-triggered activation of the HPA, direct effects of LPS on glucocorticoid release have been described. Present studies were undertaken to characterize the molecular mechanisms underlying the effect of LPS on steroid secretion in isolated rodent adrenal cells, assessing the participation of NFκB and COX-2 activities in this response. Our results show that LPS treatment stimulates steroidogenesis in murine and rat adrenocortical cells, and that Y1 cells express the binding-transducing complex TLR-4/CD14/MD-2, as demonstrated by RT-PCR. NFκB activity and COX-2 protein levels are increased in this cell line by LPS treatment, and pharmacologic and molecular manipulation of the NFκB pathway significantly affected both COX-2 protein levels and steroid production. Finally, pharmacological inhibition of COX-2 activity significantly impairs steroid production. Thus, our results strongly suggest that the mechanism involved in the stimulation of steroidogenesis by LPS in rodent adrenal cells involves the activation of the NFκB signaling pathway and the induction of COX-2. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. Thyroid cell lines in research on goitrogenesis.

    PubMed

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

  20. Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells.

    PubMed

    González, María Del Carmen; Corton, J Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Alvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

  1. Peroxisome Proliferator-Activated Receptorα Agonists Differentially Regulate Inhibitor of DNA Binding Expression in Rodents and Human Cells

    PubMed Central

    González, María del Carmen; Corton, J. Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Álvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans. PMID:22701468

  2. The thin blue line: a review and discussion of aseptic technique and postprocedural infections in rodents.

    PubMed

    Cooper, D M; McIver, R; Bianco, R

    2000-11-01

    A basic tenet of animal welfare philosophy is that pain and distress must be minimized whenever possible without interfering with the goals of the research. Aseptic technique during surgical procedures is essential to prevent pain and distress associated with post-procedural infections. However, many investigators have found that applying the aseptic techniques used for large animal and human surgery is not always practical when performing surgery on small rodents. Furthermore, the efficacy of some of these techniques for preventing post-procedural infections has been questioned. This review examines what is known about the development of postprocedural infections in animals and humans and the methods used to prevent them. Detection of postprocedural infections in rodents can be difficult unless objective measurements of physiologic indices are made. These measurements should be used experimentally to assess the relative benefits of various methods for preventing postprocedural infections. Measures of contamination, such as quantitative bacterial cultures, also can be used; however, they do not reliably predict infection rates. Much of the dogma about decontamination of skin and hair prior to surgery is not supported by valid experimental evidence. Hair removal may not be necessary. Alcohol may in fact be a better disinfectant than is often credited. Draping should be used when it contributes to the maintenance of the sterile field, but when it does not, modification of surgical technique may provide more protection from infection than the drape does. The contribution of surgical technique to the prevention of postprocedural infections is probably equal to that of aseptic technique. Further research needs to be done to assess various aseptic techniques for use in rodent surgery.

  3. The Therapeutic Potential of Induced Pluripotent Stem Cells After Stroke: Evidence from Rodent Models.

    PubMed

    Zents, Karlijn; Copray, Sjef

    2016-01-01

    Stroke is the second most common cause of death and the leading cause of disability in the world. About 30% of the people that are affected by stroke die within a year; 25% of the patients that survive stroke remain in need of care after a year. Therefore, stroke is a major burden for health care costs. The most common subtype is ischemic stroke. This type is characterized by a reduced and insufficient blood supply to a certain part of the brain. Despite the high prevalence of stroke, the currently used therapeutic interventions are limited. No therapies that aim to restore damaged neuronal tissue or to promote recovery are available nowadays. Transplantation of stem cell-derived cells has been investigated as a potential regenerative and protective treatment. Embryonic stem cell (ESC)-based cell therapy in rodent models of stroke has been shown to improve functional outcome. However, the clinical use of ESCs still raises ethical questions and implantation of ESC-derived cells requires continuous immunosuppression. The groundbreaking detection of induced pluripotent stem cells (iPSCs) has provided a most promising alternative. This mini-review summarizes current literature in which the potential use of iPSC-derived cells has been tested in rodent models of stroke. iPSC-based cell therapy has been demonstrated to improve motor function, decrease stroke volume, promote neurogenesis and angiogenesis and to exert immunomodulatory, anti-inflammatory effects in the brain of stroke-affected rodents.

  4. Adrenomedullin is expressed during rodent dental tissue development and promotes cell growth and mineralization.

    PubMed

    Musson, David S; McLachlan, Julia L; Sloan, Alastair J; Smith, Anthony J; Cooper, Paul R

    2010-01-06

    ADM (adrenomedullin) has pleiotropic effects, including regulation of inflammation, infection, angiogenesis, mineralized-tissue formation and development. Recently, we demonstrated up-regulation of the ADM transcript in diseased pulpal tissue while the protein is sequestered within the dentine extracellular matrix during dentinogenesis. The present study aimed to characterize ADM localization during rodent dental tissue development and determine its potential effects on dental cells. Finally, we sought to profile ADM transcript levels in adult organs and tissues to compare its expression in teeth relative to other tissues. Immunohistochemical analysis of developmental rat oral tissues indicated that, at E16 (embryonic day 16), ADM was present in dental epithelium and, by E18, ADM localized to the dental papilla and inner and outer dental epithelia. By E20, ADM was detected in secretory odontoblasts and ameloblasts and exhibited a similar expression profile to that of the key dentinogenesis signalling molecule, TGF-beta1 (transforming growth factor-beta1). Cell growth analysis in the dental MDPC-23, OD-21 and control 3T3 cell lines exposed to ADM (range 10(-15)-10(-7) M) together with EDTA-extracted DMPs (dentine matrix proteins) (range 0.00001-1000 mg/ml) containing comparable concentrations of ADM demonstrated that ADM stimulated a biphasic response in dental cell growth, comparable with that of DMPs, with peak stimulation observed at approximately 10(-11) M. For mineralization analysis, cell lines were exposed to combinations of 50 microg/ml ascorbic acid, 10 mM beta-G (beta-glycerophosphate), 10(-8) M DEX (dexamethasone) and ADM (range 10(-15)-10(-7) M). The results demonstrated that ADM could substitute for DEX to stimulate mineralization. Postnatally, multiple tissue expression profiling indicated abundant ADM levels in tongue and pulpal tissues. During oral and dental tissue development ADM initially localizes to epithelial tissue, whereas during later stages

  5. Computer simulation of the rodent spermatogonial stem cell niche.

    PubMed

    de Rooij, Dirk G; van Beek, Maria E A B

    2013-05-01

    A computer program has been developed that simulates the behavior of spermatogonial stem cells (SSCs) and their offspring inside and outside of the stem cell niche. Various parameters derived from previous morphological and cell kinetic studies have been used to set up an Excel-based computer program that simulates the proliferative activity of SSCs during the seminiferous epithelial cycle. SSCs and their offspring are depicted in a virtual piece of seminiferous tubule in which the daughter cells of self-renewing divisions of SSCs migrate away from each other, while after SSC differentiation a pair of cells is formed. Those SSC daughter cells that migrate out of the niche will very likely differentiate at their next division. Putting in physiologically acceptable parameters, the program renders numbers of spermatogonial cell types similar to those previously counted in whole mounts of seminiferous tubules. In this model, SSC numbers and numbers of differentiating cells remain constant for more than 50 virtual epithelial cycles, i.e., more than 1 yr of a mouse life and 2 yr of that of a Chinese hamster. The program can simulate various recent cell kinetic experiments and confirms, or offers alternative explanations for, the results obtained, showing its usefulness in spermatogenesis research.

  6. Effects of asbestos and man-made vitreous fibers on cell division in cultured human mesothelial cells in comparison to rodent cells.

    PubMed

    Pelin, K; Kivipensas, P; Linnainmaa, K

    1995-01-01

    We report the effects of chrysotile and crocidolite asbestos, and glass and rock wool fibers (man-made vitreous fibers, MMVF) on the induction of binucleate cells in vitro. The response of human mesothelial cells (target cells in fiber carcinogenesis) and rodent cells was compared. Human primary mesothelial cells, MeT-5A cells (an immortalized human mesothelial cell line), and rat liver epithelial (RLE) cells were exposed to asbestos and MMVF samples of similar size range. Milled glass wool, milled rock wool, and titanium dioxide were used as non-fibrous particle controls. All four fiber types caused statistically significant increases in the amount of binucleate cells in human primary mesothelial cells and MeT-5A cells (in the dose range 0.5-5.0 micrograms/cm2). Chrysotile and crocidolite asbestos were more effective (1.3-3.0-fold increases) than thin glass wool and thin rock wool fibers (1.3-2.2-fold increases). However, when the fiber doses were expressed as the number of fibers per culture area, the asbestos and MMVF appeared equally effective in human mesothelial cells. In RLE cells, chrysotile was the most potent inducer of binucleation (2.9-5.0-fold increases), but the response of the RLE cells to crocidolite, thin glass wool, and thin rock wool fibers was similar to the response of the human mesothelial cells. No statistically significant increases in the number of bi- or multinucleate cells were observed in human primary mesothelial cells or RLE cells exposed to the non-fibrous dusts. In MeT-5A cells exposed to 5 micrograms/cm2 of milled glass wool and milled rock wool, as well as in cultures exposed to 2 and 5 micrograms/cm2 of TiO2, significant increases were, however, observed. Our results show that rodent cells respond differently to mineral fibers than human cells. The results also add evidence to the suggested importance of disturbed cell division in fiber carcinogenesis.

  7. Live Imaging of Adult Neural Stem Cells in Rodents

    PubMed Central

    Ortega, Felipe; Costa, Marcos R.

    2016-01-01

    The generation of cells of the neural lineage within the brain is not restricted to early development. New neurons, oligodendrocytes, and astrocytes are produced in the adult brain throughout the entire murine life. However, despite the extensive research performed in the field of adult neurogenesis during the past years, fundamental questions regarding the cell biology of adult neural stem cells (aNSCs) remain to be uncovered. For instance, it is crucial to elucidate whether a single aNSC is capable of differentiating into all three different macroglial cell types in vivo or these distinct progenies constitute entirely separate lineages. Similarly, the cell cycle length, the time and mode of division (symmetric vs. asymmetric) that these cells undergo within their lineage progression are interesting questions under current investigation. In this sense, live imaging constitutes a valuable ally in the search of reliable answers to the previous questions. In spite of the current limitations of technology new approaches are being developed and outstanding amount of knowledge is being piled up providing interesting insights in the behavior of aNSCs. Here, we will review the state of the art of live imaging as well as the alternative models that currently offer new answers to critical questions. PMID:27013941

  8. Chapter 6. available lepidopteran insect cell lines

    USDA-ARS?s Scientific Manuscript database

    This chapter lists the known cell lines from Lepidoptera, largely based on previous compilations of insect cell lines published by W. Fred Hink. More than 320 lines from 65 species are listed. The official designation is given for each cell line as well as the species, tissue source, and, when kno...

  9. Control of cellular proliferation by modulation of oxidative phosphorylation in human and rodent fast-growing tumor cells

    SciTech Connect

    Rodriguez-Enriquez, Sara . E-mail: rodsar@mail.cardiologia.org.mx; Vital-Gonzalez, Paola A.; Flores-Rodriguez, Fanny L.; Marin-Hernandez, Alvaro; Ruiz-Azuara, Lena; Moreno-Sanchez, Rafael

    2006-09-01

    The relationship between cell proliferation and the rates of glycolysis and oxidative phosphorylation in HeLa (human) and AS-30D (rodent) tumor cells was evaluated. In glutamine plus glucose medium, both tumor lines grew optimally. Mitochondria were the predominant source of ATP in both cell types (66-75%), despite an active glycolysis. In glucose-free medium with glutamine, proliferation of both lines diminished by 30% but oxidative phosphorylation and the cytosolic ATP level increased by 50%. In glutamine-free medium with glucose, proliferation, oxidative phosphorylation and ATP concentration diminished drastically, although the cells were viable. Oligomycin, in medium with glutamine plus glucose, abolished growth of both tumor lines, indicating an essential role of mitochondrial ATP for tumor progression. The presumed mitochondrial inhibitors rhodamines 123 and 6G, and casiopeina II-gly, inhibited tumor cell proliferation and oxidative phosphorylation, but also glycolysis. In contrast, gossypol, iodoacetate and arsenite strongly blocked glycolysis; however, they did not affect tumor proliferation or mitochondrial metabolism. Growth of both tumor lines was highly sensitive to rhodamines and casiopeina II-gly, with IC{sub 5} values for HeLa cells lower than 0.5 {mu}M, whereas viability and proliferation of human lymphocytes were not affected by these drugs (IC{sub 5} > 30 {mu}M). Moreover, rhodamine 6G and casiopeina II-gly, at micromolar doses, prolonged the survival of animals bearing i.p. implanted AS-30D hepatoma. It is concluded that fast-growing tumor cells have a predominantly oxidative type of metabolism, which might be a potential therapeutic target.

  10. Lead line in rodents: an old sign of lead intoxication turned into a new method for environmental surveillance.

    PubMed

    de Figueiredo, Fellipe Augusto Tocchini; Ramos, Junia; Kawakita, Erika R Hashimoto; Bilal, Alina S; de Sousa, Frederico B; Swaim, William D; Issa, Joao P Mardegan; Gerlach, Raquel F

    2016-11-01

    The "lead line" was described by Henry Burton in 1840. Rodents are used as sentinels to monitor environmental pollution, but their teeth have not been used to determine lead. To determine whether lead deposits can be observed in the teeth of lead-exposed animals, since the gingival deposits known as "lead line" would likely have a correlate in the calcified tissue to which the gums are opposed during life. Male Wistar rats were exposed to lead in the drinking water (30 mg/L) since birth until 60 days-old. Molars and the incisors of each hemimandible were analyzed by scanning electron microscopy (SEM) on regular and backscattered electrons (BSE) mode. Elements were determined using electron dispersive spectroscopy (EDS). Clean cervical margins were observed on control teeth, as opposed to the findings of extensive deposits on lead-exposed animals, even in hemimandibles that had been exhumed after being buried for 90 days. BSE/EDS indicated that those deposits were an exogenous material compatible with lead sulfite. Presence of calcium, phosphorus, magnesium, carbon, lead, and oxygen is presented. Lead-exposed animals presented marked root resorption. The lead deposits characterized here for the first time show that the "lead line" seen in gums has a calcified tissue counterpart, that is detectable post-mortem even in animals exposed to a low dose of lead. This is likely a good method to detect undue lead exposure and will likely have wide application for pollution surveillance using sentinels.

  11. Morphological and functional maturation of Leydig cells: from rodent models to primates.

    PubMed

    Teerds, Katja J; Huhtaniemi, Ilpo T

    2015-01-01

    Leydig cells (LC) are the sites of testicular androgen production. Development of LC occurs in the testes of most mammalian species as two distinct growth phases, i.e. as fetal and pubertal/adult populations. In primates there are indications of a third neonatal growth phase. LC androgen production begins in embryonic life and is crucial for the intrauterine masculinization of the male fetal genital tract and brain, and continues until birth after which it rapidly declines. A short post-natal phase of LC activity in primates (including human) termed 'mini-puberty' precedes the period of juvenile quiescence. The adult population of LC evolves, depending on species, in mid- to late-prepuberty upon reawakening of the hypothalamic-pituitary-testicular axis, and these cells are responsible for testicular androgen production in adult life, which continues with a slight gradual decline until senescence. This review is an updated comparative analysis of the functional and morphological maturation of LC in model species with special reference to rodents and primates. Pubmed, Scopus, Web of Science and Google Scholar databases were searched between December 2012 and October 2014. Studies published in languages other than English or German were excluded, as were data in abstract form only. Studies available on primates were primarily examined and compared with available data from specific animal models with emphasis on rodents. Expression of different marker genes in rodents provides evidence that at least two distinct progenitor lineages give rise to the fetal LC (FLC) population, one arising from the coelomic epithelium and the other from specialized vascular-associated cells along the gonad-mesonephros border. There is general agreement that the formation and functioning of the FLC population in rodents is gonadotrophin-responsive but not gonadotrophin-dependent. In contrast, although there is in primates some controversy on the role of gonadotrophins in the formation of

  12. Rodent CNS neuron development: Timing of cell birth and death

    NASA Technical Reports Server (NTRS)

    Keefe, J. R.

    1984-01-01

    Data obtained from a staged series of single paired injections of tritiated thymidine to pregnant Wistar rats or C57B16/j mice on selected embryonic days and several postnatal times are reported. All injected specimens were allowed to come to term, each litter culled to six pups and specimens were sacrificed on PN28, with fixation and embedding for paraffin and plastic embedding. The results are derived from serial paraffin sections of PN28 animals exposed to autoradiographic processing and plotted with respect to heavily labelled cell nuclei present in the selected brain stem nuclei and sensory ganglia. Counts from each time sample/structure are totalled and the percentage of cells in the total labelled population/structure represented by each injection time interval plotted.

  13. Rodent CNS neuron development: Timing of cell birth and death

    NASA Technical Reports Server (NTRS)

    Keefe, J. R.

    1984-01-01

    Data obtained from a staged series of single paired injections of tritiated thymidine to pregnant Wistar rats or C57B16/j mice on selected embryonic days and several postnatal times are reported. All injected specimens were allowed to come to term, each litter culled to six pups and specimens were sacrificed on PN28, with fixation and embedding for paraffin and plastic embedding. The results are derived from serial paraffin sections of PN28 animals exposed to autoradiographic processing and plotted with respect to heavily labelled cell nuclei present in the selected brain stem nuclei and sensory ganglia. Counts from each time sample/structure are totalled and the percentage of cells in the total labelled population/structure represented by each injection time interval plotted.

  14. Brush cells of rodent gallbladder and stomach epithelia express neurofilaments.

    PubMed

    Luciano, Liliana; Groos, Stephanie; Reale, Enrico

    2003-02-01

    It has been suggested that brush cells (BCs), a distinct type of cell occurring in various epithelia of the respiratory and gastrointestinal tracts, may function as receptor cells. The major characteristics of BCs are a prominent brush border and an unusually highly ordered arrangement of cytoskeletal elements (F-actin, microtubules, and intermediate filaments). In this study we aimed to characterize the nature of the intermediate filaments in BCs by light and electron microscopic immunostaining. Gallbladder and stomach specimens from mice and rats, respectively, were fixed in various solutions, embedded either in paraffin or epoxy resin, and processed for immunodetection. Commercially available, well-characterized antibodies against neurofilaments, peripherin, and cytokeratin peptide 18 were used. The polyclonal antiserum cocktail to neurofilaments was applied as a supplement in a double-labeling procedure with anti-actin and anti-cytokeratin 18 antibodies. The results demonstrate that the BCs of both organs express two types of intermediate filaments, i.e., neurofilaments and cytokeratin 18 filaments, and that these have a compartmentalized distribution in the cytoplasm. BCs do not express peripherin. The immunodetection of intermediate filaments distinctive for mature neurons in BCs supports their putative receptor function. The co-expression of neurofilaments and cytokeratins is shown for the first time in healthy tissues.

  15. β-Cell Generation: Can Rodent Studies Be Translated to Humans?

    PubMed Central

    Carlotti, Françoise; Zaldumbide, Arnaud; Ellenbroek, Johanne H.; Spijker, H. Siebe; Hoeben, Rob C.; de Koning, Eelco J.

    2011-01-01

    β-cell replacement by allogeneic islet transplantation is a promising approach for patients with type 1 diabetes, but the shortage of organ donors requires new sources of β cells. Islet regeneration in vivo and generation of β-cells ex vivo followed by transplantation represent attractive therapeutic alternatives to restore the β-cell mass. In this paper, we discuss different postnatal cell types that have been envisaged as potential sources for future β-cell replacement therapy. The ultimate goal being translation to the clinic, a particular attention is given to the discrepancies between findings from studies performed in rodents (both ex vivo on primary cells and in vivo on animal models), when compared with clinical data and studies performed on human cells. PMID:22007286

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

    PubMed Central

    2011-01-01

    Background Approximately 60% of emerging viruses are of zoonotic origin, with three-fourths derived from wild animals. Many of these zoonotic diseases are transmitted by rodents with important information about their reservoir dynamics and pathogenesis missing. One main reason for the gap in our knowledge is the lack of adequate cell culture systems as models for the investigation of rodent-borne (robo) viruses in vitro. Therefore we established and characterized a new cell line, BVK168, using the kidney of a bank vole, Myodes glareolus, the most abundant member of the Arvicolinae trapped in Germany. Results BVK168 proved to be of epithelial morphology expressing tight junctions as well as adherence junction proteins. The BVK168 cells were analyzed for their infectability by several arbo- and robo-viruses: Vesicular stomatitis virus, vaccinia virus, cowpox virus, Sindbis virus, Pixuna virus, Usutu virus, Inkoo virus, Puumalavirus, and Borna disease virus (BDV). The cell line was susceptible for all tested viruses, and most interestingly also for the difficult to propagate BDV. Conclusion In conclusion, the newly established cell line from wildlife rodents seems to be an excellent tool for the isolation and characterization of new rodent-associated viruses and may be used as in vitro-model to study properties and pathogenesis of these agents. PMID:21729307

  17. Knowledge Gaps in Rodent Pancreas Biology: Taking Human Pluripotent Stem Cell-Derived Pancreatic Beta Cells into Our Own Hands.

    PubMed

    Santosa, Munirah Mohamad; Low, Blaise Su Jun; Pek, Nicole Min Qian; Teo, Adrian Kee Keong

    2015-01-01

    In the field of stem cell biology and diabetes, we and others seek to derive mature and functional human pancreatic β cells for disease modeling and cell replacement therapy. Traditionally, knowledge gathered from rodents is extended to human pancreas developmental biology research involving human pluripotent stem cells (hPSCs). While much has been learnt from rodent pancreas biology in the early steps toward Pdx1(+) pancreatic progenitors, much less is known about the transition toward Ngn3(+) pancreatic endocrine progenitors. Essentially, the later steps of pancreatic β cell development and maturation remain elusive to date. As a result, the most recent advances in the stem cell and diabetes field have relied upon combinatorial testing of numerous growth factors and chemical compounds in an arbitrary trial-and-error fashion to derive mature and functional human pancreatic β cells from hPSCs. Although this hit-or-miss approach appears to have made some headway in maturing human pancreatic β cells in vitro, its underlying biology is vaguely understood. Therefore, in this mini-review, we discuss some of these late-stage signaling pathways that are involved in human pancreatic β cell differentiation and highlight our current understanding of their relevance in rodent pancreas biology. Our efforts here unravel several novel signaling pathways that can be further studied to shed light on unexplored aspects of rodent pancreas biology. New investigations into these signaling pathways are expected to advance our knowledge in human pancreas developmental biology and to aid in the translation of stem cell biology in the context of diabetes treatments.

  18. Knowledge Gaps in Rodent Pancreas Biology: Taking Human Pluripotent Stem Cell-Derived Pancreatic Beta Cells into Our Own Hands

    PubMed Central

    Santosa, Munirah Mohamad; Low, Blaise Su Jun; Pek, Nicole Min Qian; Teo, Adrian Kee Keong

    2016-01-01

    In the field of stem cell biology and diabetes, we and others seek to derive mature and functional human pancreatic β cells for disease modeling and cell replacement therapy. Traditionally, knowledge gathered from rodents is extended to human pancreas developmental biology research involving human pluripotent stem cells (hPSCs). While much has been learnt from rodent pancreas biology in the early steps toward Pdx1+ pancreatic progenitors, much less is known about the transition toward Ngn3+ pancreatic endocrine progenitors. Essentially, the later steps of pancreatic β cell development and maturation remain elusive to date. As a result, the most recent advances in the stem cell and diabetes field have relied upon combinatorial testing of numerous growth factors and chemical compounds in an arbitrary trial-and-error fashion to derive mature and functional human pancreatic β cells from hPSCs. Although this hit-or-miss approach appears to have made some headway in maturing human pancreatic β cells in vitro, its underlying biology is vaguely understood. Therefore, in this mini-review, we discuss some of these late-stage signaling pathways that are involved in human pancreatic β cell differentiation and highlight our current understanding of their relevance in rodent pancreas biology. Our efforts here unravel several novel signaling pathways that can be further studied to shed light on unexplored aspects of rodent pancreas biology. New investigations into these signaling pathways are expected to advance our knowledge in human pancreas developmental biology and to aid in the translation of stem cell biology in the context of diabetes treatments. PMID:26834702

  19. Endogenous CD317/Tetherin limits replication of HIV-1 and murine leukemia virus in rodent cells and is resistant to antagonists from primate viruses.

    PubMed

    Goffinet, Christine; Schmidt, Sarah; Kern, Christian; Oberbremer, Lena; Keppler, Oliver T

    2010-11-01

    Human CD317 (BST-2/tetherin) is an intrinsic immunity factor that blocks the release of retroviruses, filoviruses, herpesviruses, and arenaviruses. It is unclear whether CD317 expressed endogenously in rodent cells has the capacity to interfere with the replication of the retroviral rodent pathogen murine leukemia virus (MLV) or, in the context of small-animal model development, contributes to the well-established late-phase restriction of human immunodeficiency virus type 1 (HIV-1). Here, we show that small interfering RNA (siRNA)-mediated knockdown of CD317 relieved a virion release restriction and markedly enhanced the egress of HIV-1, HIV-2, and simian immunodeficiency virus (SIV) in rat cells, including primary macrophages. Moreover, rodent CD317 potently inhibited MLV release, and siRNA-mediated depletion of CD317 in a mouse T-cell line resulted in the accelerated spread of MLV. Several virus-encoded antagonists have recently been reported to overcome the restriction imposed by human or monkey CD317, including HIV-1 Vpu, envelope glycoproteins of HIV-2 and Ebola virus, Kaposi's sarcoma-associated herpesvirus K5, and SIV Nef. In contrast, both rat and mouse CD317 showed a high degree of resistance to these viral antagonists. These data suggest that CD317 is a broadly acting and conserved mediator of innate control of retroviral infection and pathogenesis that restricts the release of retroviruses and lentiviruses in rodents. The high degree of resistance of the rodent CD317 restriction factors to antagonists from primate viruses has implications for HIV-1 small-animal model development and may guide the design of novel antiviral interventions.

  20. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  1. Functional cell classes and functional architecture in the early visual system of a highly visual rodent.

    PubMed

    Van Hooser, Stephen D; Heimel, J Alexander; Nelson, Sacha B

    2005-01-01

    Over the last 50 years, studies of receptive field properties in mammalian visual brain structures such as lateral geniculate nucleus (LGN) and primary visual cortex (V1) have suggested the existence of cell classes with unique functional response properties, and in visual cortex of many mammals these functional response properties show considerable spatial organization termed functional architecture. In recent years, there has been considerable interest in understanding the cellular mechanisms that underlie visual responses and plasticity in intact animals, and studies of individual neurons in brain slices have identified distinct cell classes on the basis of anatomical features, synaptic connectivity, or gene expression. However, the relationships between cell classes identified in studies of brain slices and those in the intact animal remain largely unclear. Rodents offer many advantages for investigating these relationships, as they are appropriate for a wide variety of experimental techniques and genetically modified mice are relatively easy to obtain or produce. Unfortunately, a barrier to using these animals in vision research is a lack of understanding of the relationship of rodent visual systems to the visual systems in more commonly studied mammals such as carnivores and non-human primates. Here we review recent comparative studies of functional response properties in LGN and V1 of a highly visual diurnal rodent, the gray squirrel. In the LGN, our data are consistent with the idea that all mammals have a class of LGN neurons that is sustained, another class that is transient, and a third class of more heterogeneous cells, but some response properties such as linearity of spatial summation, contrast gain, and dependence of receptive field size on eccentricity vary from species to species. In V1, the squirrel has many orientation-selective neurons, and these orientation-selective cells can be further subdivided into simple and complex cells. Despite the

  2. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

  3. Conserved phenotypic variation patterns, evolution along lines of least resistance, and departure due to selection in fossil rodents.

    PubMed

    Renaud, Sabrina; Auffray, Jean-Christophe; Michaux, Jacques

    2006-08-01

    Within a group of organisms, some morphologies are more readily generated than others due to internal developmental constraints. Such constraints can channel evolutionary changes into directions corresponding to the greatest intraspecific variation. Long-term evolutionary outputs, however, depend on the stability of these intraspecific patterns of variation over time and from the interplay between internal constraints and selective regimes. To address these questions, the relationship between the structure of phenotypic variance covariance matrices and direction of morphological evolution was investigated using teeth of fossil rodents. One lineage considered here leads to Stephanomys, a highly specialized genus characterized by a dental pattern supposedly favoring grass eating. Stephanomys evolved in the context of directional selection related to the climatic trend of global cooling causing an increasing proportion of grasslands in southwestern Europe. The initial divergence (up to approximately 6.5 mya) was channeled along the direction of greatest intraspecific variation, whereas after 6.5 mya, morphological evolution departed from the direction favored by internal constraints. This departure from the "lines of least resistance" was likely the consequence of an environmental degradation causing a selective gradient strong enough to overwhelm the constraints to phenotypic evolution. However, in a context of stabilizing selection, these constraints actually channel evolution, as exemplified by the lineage of Apodemus. This lineage retained a primitive diet and dental pattern over the last 10 myr. Limited morphological changes occurred nevertheless in accordance with the main patterns of intraspecific variation. The importance of these lines of least resistance directing long-term morphological evolution may explain parallel evolution of some dental patterns in murine evolution.

  4. Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties.

    PubMed

    Kubota, Hiroshi; Wu, Xin; Goodyear, Shaun M; Avarbock, Mary R; Brinster, Ralph L

    2011-08-01

    Previous studies suggest that exogenous factors crucial for spermatogonial stem cell (SSC) self-renewal are conserved among several mammalian species. Since glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial cell (C166) feeder layers. Proliferation of rabbit germ cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ cells colonized seminiferous tubules of immunodeficient mice, proliferated for at least 6 mo, while retaining an SSC phenotype in the testes of recipient mice, indicating that they were rabbit SSCs. This study demonstrates that GDNF is a mitogenic factor promoting self-renewal that is conserved between rodent and rabbit SSCs; with an evolutionary separation of ∼ 60 million years. These findings provide a foundation to study the mechanisms governing SSC self-renewal in nonrodent species.

  5. A Stem Cell Strategy Identifies Glycophorin C as a Major Erythrocyte Receptor for the Rodent Malaria Parasite Plasmodium berghei

    PubMed Central

    Yiangou, Loukia; Montandon, Ruddy; Modrzynska, Katarzyna; Rosen, Barry; Bushell, Wendy; Hale, Christine; Billker, Oliver; Rayner, Julian C.

    2016-01-01

    The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types. PMID:27362409

  6. Effects Of Excitotoxic Lesion With Inhaled Anesthetics On Nervous System Cells Of Rodents.

    PubMed

    Quiroz-Padilla, Maria Fernanda; Guillazo-Blanch, Gemma; Sanchez, Magdy Y; Dominguez-Sanchez, Maria Andrea; Gomez, Rosa Margaria

    2017-08-17

    Different anesthesia methods can variably influence excitotoxic lesion effects on the brain. The main purpose of this review is to identify potential differences in the toxicity to nervous system cells of two common inhalation anesthesia methods, isoflurane and sevoflurane, used in combination with an excitotoxic lesion procedure in rodents. The use of bioassays in animal models has provided the opportunity to examine the role of specific molecules and cellular interactions that underlie important aspects of neurotoxic effects relating to calcium homeostasis and apoptosis activation. Processes induced by NMDA antagonist drugs involve translocation of Bax protein to mitochondrial membranes, allowing extra-mitochondrial leakage of cytochrome c, followed by sequence of changes that ending in activation of CASP-3. The literature demonstrates that the use of these anesthetics in excitotoxic surgery increases neuroinflammation activity facilitating the effects of apoptosis and necrosis on nervous system cells, depending on the concentration and exposure duration of the anesthetic. High numbers of microglia and astrocytes and high levels of proinflammatory cytokines and caspase activation possibly mediate these inflammatory responses. However, it is necessary to continue studies in rodents to understand the effect of the use of inhaled anesthetics with excitotoxic lesions in different developmental stages, including newborns, juveniles and adults. Understanding the mechanisms of regulation of cell death during development can potentially provide tools to promote neuroprotection and eventually achieve the repair of the nervous system in pathological conditions. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Regulatory System for Stem/Progenitor Cell Niches in the Adult Rodent Pituitary

    PubMed Central

    Yoshida, Saishu; Kato, Takako; Kato, Yukio

    2016-01-01

    The anterior lobe of the pituitary gland is a master endocrine tissue composed of five types of endocrine cells. Although the turnover rate of pituitary endocrine cells is as low as about 1.6% per day, recent studies have demonstrated that Sex-determining region Y-box 2 (SOX2)+-cells exist as pituitary stem/progenitor cells in the adult anterior lobe and contribute to cell regeneration. Notably, SOX2+-pituitary stem/progenitor cells form two types of niches in this tissue: the marginal cell layer (MCL-niche) and the dense cell clusters scattering in the parenchyma (parenchymal-niche). However, little is known about the mechanisms and factors for regulating the pituitary stem/progenitor cell niches, as well as the functional differences between the two types of niches. Elucidation of the regulatory mechanisms in the niches might enable us to understand the cell regeneration system that acts in accordance with physiological demands in the adult pituitary. In this review, so as to reveal the regulatory mechanisms of the two types of niche, we summarize the regulatory factors and their roles in the adult rodent pituitary niches by focusing on three components: soluble factors, cell surface proteins and extracellular matrixes. PMID:26761002

  8. Persistent use of false myeloma cell lines.

    PubMed

    Drexler, Hans G; Matsuo, Yoshinobu; MacLeod, Roderick A E

    2003-09-01

    Multiple myeloma (MM) is a neoplasm of a terminally differentiated B-cell. Human myeloma cell lines were shown to be suitable model systems for use in various fields of the biological sciences. Within the last 20 years more than 100 cell lines have been established. So-called 'myeloma cell lines' have been previously reported and are still widely used which are in reality Epstein-Barr virus (EBV)-positive B-lymphoblastoid cell lines. The presence of the EBV-genome in residual normal B-cells provides them with a selective growth advantage after explantation. Cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. On closer examination, the use of false cell lines may be seen to invalidate a significant percentage of scientific work, or at least cast doubts on the relevance of these in vitro results to the cell type or tumor in vivo. Ultimately, use of cross-contaminated cell lines is a waste of human and material resources. Henceforth, it should be mandatory to prove the proper derivation of each new cell line by comparing DNA fingerprints or karyotypes of the patient's primary cells and the cultured cells. The availability of well characterized and authenticated bona fide MM cell lines is of great importance for the study of the biology, etiology and treatment of the disease.

  9. Stem cell-derived interneuron transplants as a treatment for schizophrenia: preclinical validation in a rodent model.

    PubMed

    Donegan, J J; Tyson, J A; Branch, S Y; Beckstead, M J; Anderson, S A; Lodge, D J

    2016-08-02

    An increasing literature suggests that schizophrenia is associated with a reduction in hippocampal interneuron function. Thus, we posit that stem cell-derived interneuron transplants may be an effective therapeutic strategy to reduce hippocampal hyperactivity and attenuate behavioral deficits in schizophrenia. Here we used a dual-reporter embryonic stem cell line to generate enriched populations of parvalbumin (PV)- or somatostatin (SST)-positive interneurons, which were transplanted into the ventral hippocampus of the methylazoxymethanol rodent model of schizophrenia. These interneuron transplants integrate within the existing circuitry, reduce hippocampal hyperactivity and normalize aberrant dopamine neuron activity. Further, interneuron transplants alleviate behaviors that model negative and cognitive symptoms, including deficits in social interaction and cognitive inflexibility. Interestingly, PV- and SST-enriched transplants produced differential effects on behavior, with PV-enriched populations effectively normalizing all the behaviors examined. These data suggest that the stem cell-derived interneuron transplants may represent a novel therapeutic strategy for schizophrenia.Molecular Psychiatry advance online publication, 2 August 2016; doi:10.1038/mp.2016.121.

  10. Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues

    PubMed Central

    Lee, Seoghyun; Sohn, Kyung-Cheol; Choi, Dae-Kyoung; Won, Minho; Park, Kyeong Ah; Ju, Sung-Kyu; Kang, Kidong; Bae, Young-Ki; Hur, Gang Min; Ro, Hyunju

    2016-01-01

    Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion. PMID:27673563

  11. Toward a more precise and informative nomenclature describing fetal and neonatal male germ cells in rodents.

    PubMed

    McCarrey, John R

    2013-08-01

    The germ cell lineages are among the best characterized of all cell lineages in mammals. This characterization includes precise nomenclature that distinguishes among numerous, often subtle, changes in function or morphology as development and differentiation of germ cells proceed to form the gametes. In male rodents, there are at least 41 distinct cell types that occur during progression through the male germ cell lineage that gives rise to spermatozoa. However, there is one period during male germ cell development-that which occurs immediately following the primordial germ cell stage and prior to the spermatogonial stage-for which the system of precise and informative cell type terminology is not adequate. Often, male germ cells during this period are referred to simply as "gonocytes." However, this term is inadequate for multiple reasons, and it is suggested here that nomenclature originally proposed in the 1970s by Hilscher et al., which employs the terms M-, T1-, and T2-prospermatogonia, is preferable. In this Minireview, the history, proper utilization, and advantages of this terminology relative to that of the term gonocytes are described.

  12. Immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells.

    PubMed

    Calvo, J R; Rafii-el-Idrissi, M; Pozo, D; Guerrero, J M

    1995-04-01

    This paper reviews the evidence that supports the hypothesis of the existence of specific binding sites for melatonin on immune cells. These binding sites have been described in human blood lymphocytes and granulocytes, and thymus, spleen, and bursa of Fabricius from different rodents and birds. The dissociation constant values of these binding sites are in the 0.1-1 nM range, suggesting that melatonin may play a physiological role in lymphocyte regulation. Moreover, melatonin binding sites appear to be modulated by guanine nucleotides. Therefore, in addition to other mechanisms described for the regulation of immune function by melatonin, a direct mechanism of regulation can be involved via binding of melatonin by immunocompetent cells.

  13. A bovine cell line that can be infected by natural sheep scrapie prions.

    PubMed

    Oelschlegel, Anja M; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schatzl, Herman; Schaetzl, Hermann; Groschup, Martin H

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  14. A Bovine Cell Line That Can Be Infected by Natural Sheep Scrapie Prions

    PubMed Central

    Oelschlegel, Anja M.; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schaetzl, Hermann; Groschup, Martin H.

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases. PMID:25565633

  15. Cell line fingerprinting using retroelement insertion polymorphism.

    PubMed

    Ustyugova, Svetlana V; Amosova, Anna L; Lebedev, Yuri B; Sverdlov, Eugene D

    2005-04-01

    Human cell lines are an indispensable tool for functional studies of living entities in their numerous manifestations starting with integral complex systems such as signal pathways and networks, regulation of gene ensembles, epigenetic factors, and finishing with pathological changes and impact of artificially introduced elements, such as various transgenes, on the behavior of the cell. Therefore, it is highly desirable to have reliable cell line identification techniques to make sure that the cell lines to be used in experiments are exactly what is expected. To this end, we developed a set of informative markers based on insertion polymorphism of human retroelements (REs). The set includes 47 pairs of PCR primers corresponding to introns of the human genes with dimorphic LINE1 (L1) and Alu insertions. Using locus-specific PCR assays, we have genotyped 10 human cell lines of various origins. For each of these cell lines, characteristic fingerprints were obtained. An estimated probability that two different cell lines possess the same marker genotype is about 10-18. Therefore, the proposed set of markers provides a reliable tool for cell line identification.

  16. Gender differences in the induction of chromosomal aberrations and gene mutations in rodent germ cells

    SciTech Connect

    Adler, Ilse-Dore; Carere, Angelo; Eichenlaub-Ritter, Ursula

    2007-05-15

    Germ cell mutagenicity testing provides experimental data to quantify genetic risk for exposed human populations. The majority of tests are performed with exposure of males, and female data are relatively rare. The reason for this paucity lies in the differences between male and female germ cell biology. Male germ cells are produced throughout reproductive life and all developmental stages can be ascertained by appropriate breeding schemes. In contrast, the female germ cell pool is limited, meiosis begins during embryogenesis and oocytes are arrested over long periods of time until maturation processes start for small numbers of oocytes during the oestrus cycle in mature females. The literature data are reviewed to point out possible gender differences of germ cells to exogenous agents such as chemicals or ionizing radiation. From the limited information, it can be concluded that male germ cells are more sensitive than female germ cells to the induction of chromosomal aberrations and gene mutations. However, exceptions are described which shed doubt on the extrapolation of experimental data from male rodents to the genetic risk of the human population. Furthermore, the female genome may be more sensitive to mutation induction during peri-conceptional stages compared to the male genome of the zygote. With few exceptions, germ cell experiments have been carried out under high acute exposure to optimize the effects and to compensate for the limited sample size in animal experiments. Human exposure to environmental agents, on the other hand, is usually chronic and involves low doses. Under these conditions, gender differences may become apparent that have not been studied so far. Additionally, data are reviewed that suggest a false impression of safety when responses are negative under high acute exposure of male rodents while a mutational response is induced by low chronic exposure. The classical (morphological) germ cell mutation tests are not performed anymore

  17. Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells

    PubMed Central

    Novobrantseva, Tatiana I; Borodovsky, Anna; Wong, Jamie; Klebanov, Boris; Zafari, Mohammad; Yucius, Kristina; Querbes, William; Ge, Pei; Ruda, Vera M; Milstein, Stuart; Speciner, Lauren; Duncan, Rick; Barros, Scott; Basha, Genc; Cullis, Pieter; Akinc, Akin; Donahoe, Jessica S; Narayanannair Jayaprakash, K; Jayaraman, Muthusamy; Bogorad, Roman L; Love, Kevin; Whitehead, Katie; Levins, Chris; Manoharan, Muthiah; Swirski, Filip K; Weissleder, Ralph; Langer, Robert; Anderson, Daniel G; de Fougerolles, Antonin; Nahrendorf, Matthias; Koteliansky, Victor

    2012-01-01

    Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells. PMID:23344621

  18. Rodent Control

    ERIC Educational Resources Information Center

    Indian Journal of Adult Education, 1975

    1975-01-01

    Strategies for rodent control in crop fields, threshing yards, and rural residential areas are presented together with an operational plan for implementing a program for rodent control at the national level. Training personnel in rodent control procedures and procedures for educating the public in the necessity for control are covered. (EC)

  19. Rodent Control

    ERIC Educational Resources Information Center

    Indian Journal of Adult Education, 1975

    1975-01-01

    Strategies for rodent control in crop fields, threshing yards, and rural residential areas are presented together with an operational plan for implementing a program for rodent control at the national level. Training personnel in rodent control procedures and procedures for educating the public in the necessity for control are covered. (EC)

  20. RODENT AND HUMAN NEUROPROGENITOR CELLS FOR HIGH-CONTENT SCREENS OF CHEMICAL EFFECTS ON PROLIFERATION AND APOPTOSIS

    EPA Science Inventory

    The objective of these experiments is to develop high-throughput screens for proliferation and apoptosis in order to compare rodent and human neuroprogenitor cell responses to potential developmental neurotoxicants. Effects of 4 chemicals on proliferation and apoptosis in mouse c...

  1. RODENT AND HUMAN NEUROPROGENITOR CELLS FOR HIGH-CONTENT SCREENS OF CHEMICAL EFFECTS ON PROLIFERATION AND APOPTOSIS

    EPA Science Inventory

    The objective of these experiments is to develop high-throughput screens for proliferation and apoptosis in order to compare rodent and human neuroprogenitor cell responses to potential developmental neurotoxicants. Effects of 4 chemicals on proliferation and apoptosis in mouse c...

  2. Early and late B cell immune responses in lethal and self-cured rodent malaria.

    PubMed

    Azcárate, Isabel G; Marín-García, Patricia; Pérez-Benavente, Susana; Diez, Amalia; Puyet, Antonio; Bautista, José M

    2015-05-01

    ICR mice have heterogeneous susceptibility to lethal Plasmodium yoelii yoelii 17XL from the first days of experimental infection as evidenced by the different parasitemia levels and clinical outcomes. This mouse model has revealed specific immune responses on peripheral blood correlating with the infection fate of the animals. To search for immune-markers linked to parasitemia we examined B lymphocytes in organs of the immune system as key effectors of rodent immunity against malaria. To determine changes in immune cellularity fostered by the different prognostic parasitemia we examined B cell subsets in low (<15%) and high (>50%) parasitized mice during the first days of the infection. In the case of surviving mice, we studied the preservation of memory immune response 500 days after the primary P. yoelii challenge. Correlating with the parasitemia level, it was observed an increase in total cellularity of spleen during the first week of infection which remained after 16 months of the infection in surviving animals. B cell subsets were also modified across the different infection fates. Subpopulation as follicular B cells and B-1 cells proportions behaved differently depending on the parasitemia kinetics. In addition, peritoneal cavity cells proliferated in response to high parasitemia. More significantly, P. yoelii -specific memory B cells remained in the spleen 500 days after the primo-infection. This study demonstrates that B cell kinetics is influenced by the different parasitemia courses which are naturally developed within a same strain of untreated mice. We show that high levels of parasitemia at the beginning of infection promote an extremely fast and exacerbate response of several cell populations in spleen and peritoneal cavity that, in addition, do not follow the kinetics observed in peripheral blood. Furthermore, our results describe the longest persistence of memory B cells long time upon a single malaria infection in mice.

  3. CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria.

    PubMed

    Lau, Lei Shong; Fernandez-Ruiz, Daniel; Mollard, Vanessa; Sturm, Angelika; Neller, Michelle A; Cozijnsen, Anton; Gregory, Julia L; Davey, Gayle M; Jones, Claerwen M; Lin, Yi-Hsuan; Haque, Ashraful; Engwerda, Christian R; Nie, Catherine Q; Hansen, Diana S; Murphy, Kenneth M; Papenfuss, Anthony T; Miles, John J; Burrows, Scott R; de Koning-Ward, Tania; McFadden, Geoffrey I; Carbone, Francis R; Crabb, Brendan S; Heath, William R

    2014-05-01

    To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

  4. CD8+ T Cells from a Novel T Cell Receptor Transgenic Mouse Induce Liver-Stage Immunity That Can Be Boosted by Blood-Stage Infection in Rodent Malaria

    PubMed Central

    Mollard, Vanessa; Sturm, Angelika; Neller, Michelle A.; Cozijnsen, Anton; Gregory, Julia L.; Davey, Gayle M.; Jones, Claerwen M.; Lin, Yi-Hsuan; Haque, Ashraful; Engwerda, Christian R.; Nie, Catherine Q.; Hansen, Diana S.; Murphy, Kenneth M.; Papenfuss, Anthony T.; Miles, John J.; Burrows, Scott R.; de Koning-Ward, Tania; McFadden, Geoffrey I.; Carbone, Francis R.; Crabb, Brendan S.; Heath, William R.

    2014-01-01

    To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections. PMID:24854165

  5. Standards for Cell Line Authentication and Beyond

    PubMed Central

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  6. Anticonvulsant drug-induced cell death in the developing white matter of the rodent brain.

    PubMed

    Kaushal, Suhasini; Tamer, Zenab; Opoku, Freda; Forcelli, Patrick A

    2016-05-01

    During critical periods of brain development, both seizures and anticonvulsant medications can affect neurodevelopmental outcomes. In rodent models, many anticonvulsants trigger neuronal apoptosis. However, white matter apoptosis (WMA) has not been examined after anticonvulsant drug treatment. Herein, we sought to determine if anticonvulsant drugs induced apoptosis in the developing white matter (WM) in a rodent model. Postnatal day (P)7 rats were treated with phenobarbital (PB-75), MK-801 (dizocilpine, 0.5), lamotrigine (LTG-20), carbamazepine (CBZ-100), phenytoin (PHT-50), levetiracetam (LEV-250), or saline; all doses are mg/kg. Brain tissue collected 24 h after treatment was stained using the terminal deoxynucleotidyl transferase dUTP nick end labeling method. The number of degenerating cells within WM, that is, anterior commissure (AC), corpus callosum, cingulum, and hippocampus-associated WM tracts, was quantified. Saline-treated rats showed low baseline level of apoptosis in developing WM on P8 in all the areas examined. PB, PHT, and MK-801 significantly increased apoptosis in all four brain areas examined. Exposure to CBZ, LTG, or LEV failed to increase apoptosis in all regions. Commonly used anticonvulsants (PB, PHT) cause apoptosis in the developing WM in a rat model; the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 has a similar effect. These results are consistent with reports of anesthesia-induced WMA during brain development. Consistent with the lack of neuronal apoptosis caused by LTG, LEV, and CBZ, these drugs did not cause WMA. Many infants treated with anticonvulsant drugs have underlying neurologic injury, including WM damage (e.g., following intraventricular hemorrhage [IVH] or hypoxic-ischemic encephalopathy [HIE]). The degree to which anticonvulsant drug treatment will alter outcomes in the presence of underlying injury remains to be examined, but avoiding drugs (when possible) that induce WMA may be beneficial. Wiley Periodicals, Inc

  7. Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines

    PubMed Central

    Sultan, Serageldeen; Lan, Nguyen Thi; Ueda, Toshiki; Yamaguchi, Ryoji; Maeda, Ken; Kai, Kazushige

    2009-01-01

    Backgrounds The aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. Methods The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST) cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains) and by observing the development of cytopathic effect (CPE) in infected cultures of hamster cell lines. Results Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. Conclusion The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically. PMID:19835588

  8. Embryonic stem cell lines of nonhuman primates.

    PubMed

    Nakatsuji, Norio; Suemori, Hirofumi

    2002-06-26

    Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics. Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases. So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice. The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is

  9. Thyroid cancer cell lines: an overview

    PubMed Central

    Saiselet, Manuel; Floor, Sébastien; Tarabichi, Maxime; Dom, Geneviève; Hébrant, Aline; van Staveren, Wilma C. G.; Maenhaut, Carine

    2012-01-01

    Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during

  10. Tolerogenic dendritic cells and negative vaccination in transplantation: from rodents to clinical trials

    PubMed Central

    Moreau, Aurélie; Varey, Emilie; Bériou, Gaëlle; Hill, Marcelo; Bouchet-Delbos, Laurence; Segovia, Mercedes; Cuturi, Maria-Cristina

    2012-01-01

    The use of immunosuppressive (IS) drugs to treat transplant recipients has markedly reduced the incidence of acute rejection and early graft loss. However, such treatments have numerous adverse side effects and fail to prevent chronic allograft dysfunction. In this context, therapies based on the adoptive transfer of regulatory cells are promising strategies to induce indefinite transplant survival. The use of tolerogenic dendritic cells (DC) has shown great potential, as preliminary experiments in rodents have demonstrated that administration of tolerogenic DC prolongs graft survival. Recipient DC, Donor DC, or Donor Ag-pulsed recipient DC have been used in preclinical studies and administration of these cells with suboptimal immunosuppression increases their tolerogenic potential. We have demonstrated that autologous unpulsed tolerogenic DC injected in the presence of suboptimal immunosuppression are able to induce Ag-specific allograft tolerance. We derived similar tolerogenic DC in different animal models (mice and non-human primates) and confirmed their protective abilities in vitro and in vivo. The mechanisms involved in the tolerance induced by autologous tolerogenic DC were also investigated. With the aim of using autologous DC in kidney transplant patients, we have developed and characterized tolerogenic monocyte-derived DC in humans. In this review, we will discuss the preclinical studies and describe our recent results from the generation and characterization of tolerogenic monocyte-derived DC in humans for a clinical application. We will also discuss the limits and difficulties in translating preclinical experiments to theclinic. PMID:22908013

  11. Embryonic stem cells can be used to construct hybrid cell lines containing a single, selectable murine chromosome.

    PubMed

    Jakobs, P M; Smith, L; Thayer, M; Grompe, M

    1999-04-01

    Microcell-mediated chromosome transfer is a useful technique for the study of gene function, gene regulation, gene mapping, and functional cloning in mammalian cells. Complete panels of donor cell lines, each containing a different human chromosome, have been developed. These donor cell lines contain a single human chromosome marked with a dominant selectable gene in a rodent cell background. However, a similar panel does not exist for murine chromosomes. To produce mouse monochromosomal donor hybrids, we have utilized embryonic stem (ES) cells with targeted gene disruptions of known chromosomal location as starting material. ES cells with mutations in aprt, fyn, and myc were utilized to generate monochromosomal hybrids with neomycin phosphotransferase-marked murine Chr 8, 10, or 15 respectively in a hamster or rat background. This same methodology can be used to generate a complete panel of marked mouse chromosomes for somatic cell genetic experimentaion.

  12. Scrapie infection in experimental rodents and SMB-S15 cells decreased the brain endogenous levels and activities of Sirt1.

    PubMed

    Wang, Jing; Zhang, Jin; Shi, Qi; Zhang, Bao-Yun; Chen, Cao; Chen, Li-Na; Sun, Jing; Wang, Hui; Xiao, Kang; Dong, Xiao-Ping

    2015-04-01

    Prion diseases are composed of a group of fatal neurodegenerative disorders resulting from misfolding of cellular prion (PrP(C)) into scrapie prion (PrP(Sc)). Sirt1, a class III histone deacetylase, has been reported to protect neuronal cells against PrP (106-126)-induced cell death. To address the potential role of Sirt1 during prion infection, the levels and enzyme activities of Sirt1 in the brains of scrapie-infected rodents, including hamsters infected with strain 263K, mice infected with strains 139A and ME7, and in prion infected SMB-S15 cells, were analyzed. Western blots revealed that endogenous Sirt1 levels were significantly decreased in all tested scrapie-infected models. Dynamic assays of brain Sirt1 levels in 263K-infected hamsters during incubation period showed a time-dependent decrease. The acetylating forms of Sirt1 target proteins, P53, PGC-1, and STAT3, markedly increased both in the brains of scrapie-infected rodents and in SMB-S15 cells, representing decreased Sirt1 activity. Immunofluorescent assays illustrated that Sirt1 predominately localized in cytosol of SMB-S15 cells but clearly distributed in nucleus of its normal partner cell line, SMB-PS. Moreover, accompanying with increase of Sirt1 level and decrease of acetyl-P53 level, treatments with Sirt1 activators SRT1720 and resveratrol in SMB-S15 cells significantly reduced PrP(Sc); at the same time, the cellular distribution of PrP proteins became normal, and the cell proliferating state was slightly improved. These data indicate that prion infection notably attenuates the Sirt1 activity in host cells. Sensitivity of the PrP(Sc) to Sirt1 activators highlights a potential role of Sirt1 in prion therapeutics.

  13. Genes amplified and overexpressed in human multidrug-resistant cell lines.

    PubMed

    Van der Bliek, A M; Baas, F; Van der Velde-Koerts, T; Biedler, J L; Meyers, M B; Ozols, R F; Hamilton, T C; Joenje, H; Borst, P

    1988-11-01

    Multidrug resistance (MDR) is associated with overproduction of Mr 170,000 membrane proteins (P-glycoproteins) caused by either gene amplification, transcriptional activation, or both. In rodents the amplified domain comprises genes that encode P-glycoproteins and at least five unrelated genes, one of which encodes the calcium-binding protein sorcin. The amplification and increased expression of these genes always includes one P-glycoprotein-encoding gene (pgp1 in hamsters, homologous to mdr1 in humans). In human MDR cells only elevated mdr1 expression has been shown thusfar, although another P-glycoprotein encoding gene (mdr3, homologous to hamster pgp3) is closely linked. Here we show that the human homolog of the hamster sorcin gene resides on chromosome 7 like the P-glycoprotein-encoding genes. Furthermore, gene classes designated 4, 5, and 6 are coamplified with mdr1 and mdr3 in the human ovarian carcinoma cell line 2780AD, which strongly suggests that the overall structure of the human MDR domain is the same as in rodents. Class 6 was moderately and mdr1 was highly overexpressed in this cell line. Four other human MDR cell lines also have much higher mdr1 overexpression than expected from the relatively low levels (2- to 30-fold) of gene amplification. This contrasts with the results of previous work with rodent MDR cells, in which the increase in P-glycoprotein mRNA levels usually parallels the increase in gene copy number. Although four of the five human MDR cell lines have coamplified mdr3, its expression was undetectable. Our results confirm the central role of the mdr1 (pgp1) gene in MDR and suggest that different cross-resistance patterns are not due to differential expression of different P-glycoprotein genes.

  14. Study of glucose uptake activity of Helicteres isora Linn. fruits in L-6 cell lines

    PubMed Central

    Gupta, R. N.; Pareek, Anil; Suthar, Manish; Rathore, Garvendra S.; Basniwal, Pawan K.; Jain, Deepti

    2009-01-01

    The effect of hot water extract of fruits of Helicteres isora on glucose uptake was studied in rodent skeletal muscle cells (L-6 cells) involved in glucose utilization. H. isora is an antidiabetic medicinal plant being used in Indian traditional medicine. Hot water extracts were analysed for glucose uptake activity and found to be significantly active at 200 μg/ml dose comparable with insulin and metformin. Elevation of glucose uptake by H. isora in association with glucose transport supported the upregulation of glucose uptake. It was concluded that hot water extract of H. isora activate glucose uptake in L-6 cell line of mouse skeletal muscles. PMID:20336200

  15. Study of glucose uptake activity of Helicteres isora Linn. fruits in L-6 cell lines.

    PubMed

    Gupta, R N; Pareek, Anil; Suthar, Manish; Rathore, Garvendra S; Basniwal, Pawan K; Jain, Deepti

    2009-10-01

    The effect of hot water extract of fruits of Helicteres isora on glucose uptake was studied in rodent skeletal muscle cells (L-6 cells) involved in glucose utilization. H. isora is an antidiabetic medicinal plant being used in Indian traditional medicine. Hot water extracts were analysed for glucose uptake activity and found to be significantly active at 200 mug/ml dose comparable with insulin and metformin. Elevation of glucose uptake by H. isora in association with glucose transport supported the upregulation of glucose uptake. It was concluded that hot water extract of H. isora activate glucose uptake in L-6 cell line of mouse skeletal muscles.

  16. The quantification of testicular cells during the postnatal development in two Caviomorph rodents: the guinea pig (Cavia porcellus) and the cutia (Dasyprocta agouti).

    PubMed

    Simões, Luciana S; Sasahara, Tais H C; Favaron, Phelipe O; Marques, Ricardo; Oliveira, Moacir F DE; Machado, Márcia R F; Miglino, Maria Angelica

    2017-01-01

    The germinative, Sertoli and Leydig cells of two caviomorph rodents (Cavia porcellus and Dasyprocta agouti) were counted as well as the estimation of the total volume of the testis and the total volume of seminiferous tubules and interstitium in prepubertal, pubertal and adult animals. The number of spermatogonia, spermatocytes and spermatids cells increased during the pubertal phase in both rodents, notably the spermatid cells. The spermatocyte and spermatid slightly decreased in the adult of both rodents, but the increment in spermatogonia cells number was seen, mainly in cutias. The number of Sertoli cells increased in pubertal rodents, but in the adult the number reduced. Substantial number of Leydig cells was counted in pubertal and adult guinea pigs. In cutias, the number of Leydig cells increased in pubertal phase and decline in adults. The design-based stereological method has proven to be unbiased and reliable to be applied in reproduction studies.

  17. Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumors in rodents.

    PubMed Central

    Hurley, P M

    1998-01-01

    Of 240 pesticides screened for carcinogenicity by the U.S. Environmental Protection Agency Office of Pesticide Programs, at least 24 (10%) produce thyroid follicular cell tumors in rodents. Thirteen of the thyroid carcinogens also induce liver tumors, mainly in mice, and 9 chemicals produce tumors at other sites. Some mutagenic data are available on all 24 pesticides producing thyroid tumors. Mutagenicity does not seem to be a major determinant in thyroid carcinogenicity, except for possibly acetochlor; evidence is less convincing for ethylene thiourea and etridiazole. Studies on thyroid-pituitary functioning, including indications of thyroid cell growth and/or changes in thyroxine, triiodothyronine, or thyroid-stimulating hormone levels, are available on 19 pesticides. No such antithyroid information is available for etridiazole, N-octyl bicycloheptene dicarboximide, terbutryn, triadimefon, and trifluralin. Of the studied chemicals, only bromacil lacks antithyroid activity under study conditions. Intrathyroidal and extrathyroidal sites of action are found: amitrole, ethylene thiourea, and mancozeb are thyroid peroxidase inhibitors; and acetochlor, clofentezine, fenbuconazole, fipronil, pendimethalin, pentachloronitrobenzene, prodiamine, pyrimethanil, and thiazopyr seem to enhance the hepatic metabolism and excretion of thyroid hormone. Thus, with 12 pesticides that mode of action judgments can be made, 11 disrupt thyroid-pituitary homeostasis only; no chemical is mutagenic only; and acetochlor may have both antithyroid and some mutagenic activity. More information is needed to identify other potential antithyroid modes of thyroid carcinogenic action. PMID:9681970

  18. Satellite glial cells in dorsal root ganglia are activated in streptozotocin-treated rodents.

    PubMed

    Hanani, Menachem; Blum, Erez; Liu, Shuangmei; Peng, Lichao; Liang, Shangdong

    2014-12-01

    Neuropathic pain is a very common complication in diabetes mellitus (DM), and treatment for it is limited. As DM is becoming a global epidemic it is important to understand and treat this problem. The mechanisms of diabetic neuropathic pain are largely obscure. Recent studies have shown that glial cells are important for a variety of neuropathic pain types, and we investigated what are the changes that satellite glial cells (SGCs) in dorsal root ganglia undergo in a DM type 1 model, induced by streptozotocin (STZ) in mice and rats. We carried out immunohistochemical studies to learn about changes in the activation marker glial fibrillary acidic protein (GFAP) in SGCs. We found that after STZ-treatment the number of neurons surrounded with GFAP-positive SGCs in dorsal root ganglia increased 4-fold in mice and 5-fold in rats. Western blotting for GFAP, which was done only on rats because of the larger size of the ganglia, showed an increase of about 2-fold in STZ-treated rats, supporting the immunohistochemical results. These results indicate for the first time that SGCs are activated in rodent models of DM1. As SGC activation appears to contribute to chronic pain, these results suggest that SGCs may participate in the generation and maintenance of diabetic neuropathic pain, and can serve as a potential therapeutic target.

  19. Prostaglandin Actions in Established Insect Cell Lines

    USDA-ARS?s Scientific Manuscript database

    Prostaglandins (PGs) are oxygenated metabolites of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids that serve as biochemical signals that mediate a wide range of physiological functions in animal cells. For example, PGs influence protein expression in establish insect cell lines ...

  20. Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

    PubMed Central

    Iandiev, Ianors; Wurm, Antje; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Robson, Simon C.; Zimmermann, Herbert

    2007-01-01

    Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y1 and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y1, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume. PMID:18404455

  1. Virus Discovery Using Tick Cell Lines

    PubMed Central

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  2. Use of the Syrian hamster embryo cell transformation assay for carcinogenicity prediction of chemical currently being tested by the National Toxicology Program in rodent bioassays

    SciTech Connect

    Kerckaert, G.A.; LeBoeuf, R.A.; Isfort, R.J.; Brauninger, R.

    1996-10-01

    The Syrian hamster embryo (SHE) cell transformation assay was used to predict the carcinogenicity of 26 chemicals currently being tested in the rodent bioassay by the National Toxicology Program as part of its program titled {open_quotes}Strategies for Predicting Chemical Carcinogenesis in Rodents.{close_quotes} Of these 26 chemicals, 17 were found to be positive in the SHE cell transformation assay while 9 were negative. Carcinogenicity predictions were made for these chemicals, based upon the SHE cell transformation assay results. Our predictions will be compared with the rodent bioassay results as they become available. 11 refs., 2 tabs.

  3. Mushroom Polysaccharides: Chemistry and Antiobesity, Antidiabetes, Anticancer, and Antibiotic Properties in Cells, Rodents, and Humans

    PubMed Central

    Friedman, Mendel

    2016-01-01

    More than 2000 species of edible and/or medicinal mushrooms have been identified to date, many of which are widely consumed, stimulating much research on their health-promoting properties. These properties are associated with bioactive compounds produced by the mushrooms, including polysaccharides. Although β-glucans (homopolysaccharides) are believed to be the major bioactive polysaccharides of mushrooms, other types of mushroom polysaccharides (heteropolysaccharides) also possess biological properties. Here we survey the chemistry of such health-promoting polysaccharides and their reported antiobesity and antidiabetic properties as well as selected anticarcinogenic, antimicrobial, and antiviral effects that demonstrate their multiple health-promoting potential. The associated antioxidative, anti-inflammatory, and immunomodulating activities in fat cells, rodents, and humans are also discussed. The mechanisms of action involve the gut microbiota, meaning the polysaccharides act as prebiotics in the digestive system. Also covered here are the nutritional, functional food, clinical, and epidemiological studies designed to assess the health-promoting properties of polysaccharides, individually and as blended mixtures, against obesity, diabetes, cancer, and infectious diseases, and suggestions for further research. The collated information and suggested research needs might guide further studies needed for a better understanding of the health-promoting properties of mushroom polysaccharides and enhance their use to help prevent and treat human chronic diseases. PMID:28231175

  4. Mushroom Polysaccharides: Chemistry and Antiobesity, Antidiabetes, Anticancer, and Antibiotic Properties in Cells, Rodents, and Humans.

    PubMed

    Friedman, Mendel

    2016-11-29

    More than 2000 species of edible and/or medicinal mushrooms have been identified to date, many of which are widely consumed, stimulating much research on their health-promoting properties. These properties are associated with bioactive compounds produced by the mushrooms, including polysaccharides. Although β-glucans (homopolysaccharides) are believed to be the major bioactive polysaccharides of mushrooms, other types of mushroom polysaccharides (heteropolysaccharides) also possess biological properties. Here we survey the chemistry of such health-promoting polysaccharides and their reported antiobesity and antidiabetic properties as well as selected anticarcinogenic, antimicrobial, and antiviral effects that demonstrate their multiple health-promoting potential. The associated antioxidative, anti-inflammatory, and immunomodulating activities in fat cells, rodents, and humans are also discussed. The mechanisms of action involve the gut microbiota, meaning the polysaccharides act as prebiotics in the digestive system. Also covered here are the nutritional, functional food, clinical, and epidemiological studies designed to assess the health-promoting properties of polysaccharides, individually and as blended mixtures, against obesity, diabetes, cancer, and infectious diseases, and suggestions for further research. The collated information and suggested research needs might guide further studies needed for a better understanding of the health-promoting properties of mushroom polysaccharides and enhance their use to help prevent and treat human chronic diseases.

  5. The novel, actin-like protein Tact3 is expressed in rodent testicular haploid germ cells.

    PubMed

    Oh, Sung-Dug; Park, Soo-Yun; Park, Jae-Il; Chun, Sang-Young; Ryu, Tae-hun; Soh, Jaemog

    2013-12-01

    Mouse testis actin-like proteins 1 and 2 (mTact1 and mTact2), which are expressed in murine haploid germ cells, have been described previously. Here, we report the cloning and characterization of a third actin-like protein from rat, rat testis actin-like protein 3 (rTact3). The complete cDNA of the rTact3 gene was approximately 3.7 kb in length, and its corresponding amino acid sequence consisted of 1219 amino acids. The rTact3 gene lacks introns, similar to mTact1 and mTact2. The 356 C-terminal amino acids of rTact3 showed 43% homology with mTact1, whereas the 863 N-terminal amino acids did not show any significant homology. Northern blot analysis revealed that rTact3 mRNA was expressed only in adult rat testes and not during the prepubescent stage. In situ hybridization revealed that rTact3 was expressed exclusively during round and elongated spermatids maturation stages in rat testes. Immunohistochemical experiments using antibodies raised against a synthetic peptide showed that the expression of the rTact3 protein was also restricted in round and elongated spermatids, specifically in the head and acrosome of mature rat sperm. The 5′-flanking region of the mTact3 gene was found to contain a TATA-box motif as well as two putative CREB/c-Jun and five C/EBP motifs. mTact3 promoter activity was enhanced in a dose-dependent manner by the transfection of CREB, c-Jun, or C/EBP in NIH3T3 cells. These results suggest that Tact3 proteins might play an important role in rodent germ-cell development. © 2013 Wiley Periodicals, Inc.

  6. Time-Lapse Imaging of Red Blood Cell Invasion by the Rodent Malaria Parasite Plasmodium yoelii

    PubMed Central

    Yahata, Kazuhide; Treeck, Moritz; Culleton, Richard; Gilberger, Tim-Wolf; Kaneko, Osamu

    2012-01-01

    In order to propagate within the mammalian host, malaria parasites must invade red blood cells (RBCs). This process offers a window of opportunity in which to target the parasite with drugs or vaccines. However, most of the studies relating to RBC invasion have analyzed the molecular interactions of parasite proteins with host cells under static conditions, and the dynamics of these interactions remain largely unstudied. Time-lapse imaging of RBC invasion is a powerful technique to investigate cell invasion and has been reported for Plasmodium knowlesi and Plasmodium falciparum. However, experimental modification of genetic loci is laborious and time consuming for these species. We have established a system of time-lapse imaging for the rodent malaria parasite Plasmodium yoelii, for which modification of genetic loci is quicker and simpler. We compared the kinetics of RBC invasion by P. yoelii with that of P. falciparum and found that the overall kinetics during invasion were similar, with some exceptions. The most striking of these differences is that, following egress from the RBC, the shape of P. yoelii merozoites gradually changes from flat elongated ovals to spherical bodies, a process taking about 60 sec. During this period merozoites were able to attach to and deform the RBC membrane, but were not able to reorient and invade. We propose that this morphological change of P. yoelii merozoites may be related to the secretion or activation of invasion-related proteins. Thus the P. yoelii merozoite appears to be an excellent model to analyze the molecular dynamics of RBC invasion, particularly during the morphological transition phase, which could serve as an expanded window that cannot be observed in P. falciparum. PMID:23227208

  7. STR DNA typing of human cell lines: detection of intra- and interspecies cross-contamination.

    PubMed

    Dirks, Wilhelm G; Drexler, Hans G

    2013-01-01

    Inter- and intraspecies cross-contaminations (CCs) of human and animal cells represent a chronic problem in cell cultures leading to false data. Microsatellite loci in the human genome harboring short tandem repeat (STR) DNA markers allow individualization of cell lines at the DNA level. Thus, fluorescence polymerase chain reaction amplification of STR loci D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX, CSF1PO, and Amelogenin for gender determination is the gold standard for authentication of human cell lines and represents an international reference technique. The major cell banks of the USA, Germany, and Japan (ATCC, DSMZ, JCRB, and RIKEN, respectively) have built compatible STR databases to ensure the availability of STR reference profiles. Upon determination of an STR profile of a human cell line, the suspected identity can be proven by online verification of customer-made STR data sets on the homepage of the DSMZ institute. Furthermore, an additional tetraplex PCR has been established to detect mitochondrial DNA sequences of rodent cells within a human cell culture population. Since authentic cell lines are the main prerequisite for rational research and biotechnology, the next sections describe a rapid and reliable method available to students, technicians, and scientists for certifying identity and purity of human cell lines of interest.

  8. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

    Apparatus for processing a metallic fluid containing iron oxide, container for a molten metal including an electrically conductive refractory disposed for contact with the molten metal which contains iron oxide, an electrolyte in the form of a basic slag on top of the molten metal, an electrode in the container in contcat with the slag electrically separated from the refractory, and means for establishing a voltage across the refractory and the electrode to reduce iron oxide to iron at the surface of the refractory in contact with the iron oxide containing fluid. A process is disclosed for refining an iron product containing not more than about 10% by weight oxygen and not more than about 10% by weight sulfur, comprising providing an electrolyte of a slag containing one or more of calcium oxide, magnesium oxide, silica or alumina, providing a cathode of the iron product in contact with the electrolyte, providing an anode in contact with the electrolyte electrically separated from the cathode, and operating an electrochemical cell formed by the anode, the cathode and the electrolyte to separate oxygen or sulfur present in the iron product therefrom.

  9. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    PubMed

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  10. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines

    PubMed Central

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines. PMID:18927105

  11. A simple method to obtain pure cultures of multiciliated ependymal cells from adult rodents.

    PubMed

    Grondona, J M; Granados-Durán, P; Fernández-Llebrez, P; López-Ávalos, M D

    2013-01-01

    Ependymal cells form an epithelium lining the ventricular cavities of the vertebrate brain. Numerous methods to obtain primary culture ependymal cells have been developed. Most of them use foetal or neonatal rat brain and the few that utilize adult brain hardly achieve purity. Here, we describe a simple and novel method to obtain a pure non-adherent ependymal cell culture from explants of the striatal and septal walls of the lateral ventricles. The combination of a low incubation temperature followed by a gentle enzymatic digestion allows the detachment of most of the ependymal cells from the ventricular wall in a period of 6 h. Along with ependymal cells, a low percentage (less than 6 %) of non-ependymal cells also detaches. However, they do not survive under two restrictive culture conditions: (1) a simple medium (alpha-MEM with glucose) without any supplement; and (2) a low density of 1 cell/µl. This purification method strategy does not require cell labelling with antibodies and cell sorting, which makes it a simpler and cheaper procedure than other methods previously described. After a period of 48 h, only ependymal cells survive such conditions, revealing the remarkable survival capacity of ependymal cells. Ependymal cells can be maintained in culture for up to 7-10 days, with the best survival rates obtained in Neurobasal supplemented with B27 among the tested media. After 7 days in culture, ependymal cells lose most of the cilia and therefore the mobility, while acquiring radial glial cell markers (GFAP, BLBP, GLAST). This interesting fact might indicate a reprogramming of the cell identity.

  12. Characterization of GABA- and glycine-induced currents of solitary rodent retinal ganglion cells in culture.

    PubMed

    Tauck, D L; Frosch, M P; Lipton, S A

    1988-10-01

    Ganglion cells were fluorescently labeled, dissociated from 7- to 11-day-old rodent retinas, and placed in tissue culture. Whole-cell recordings with patch electrodes were obtained from solitary cells lacking processes, which permitted a high-quality space clamp. Both GABA (1-200 microM) and glycine (10-300 microM) produced large increases in membrane conductance in virtually every ganglion cell tested, including ganglion cells from different size classes in both rats and mice. Taurine evoked responses similar to those of glycine, but considerably greater concentrations of taurine (150-300 microM) were necessary to observe any effect. Since 20 microM GABA produced approximately the same response as 100 microM glycine, the effects of these two concentrations were compared under various conditions. When recording with chloride distributed equally across the membrane, the reversal potential of the agonist-induced currents was approximately 0 mV. When the internal chloride was reduced by substitution with aspartate, the reversal potential shifted in a negative direction by about 42 mV, indicating that the current was carried mainly by chloride ions. Strychnine (1-5 microM) completely and reversibly blocked the actions of glycine (100 microM) but not those of GABA (20 microM); however, higher concentrations of strychnine (20 microM) nearly totally inhibited the current elicited by GABA (20 microM). The responses to glycine (100 microM) were not affected by bicuculline methiodide (20 microM) or picrotoxinin (20 microM). In contrast, bicuculline methiodide (10 microM) and picrotoxinin (10 microM) reversibly blocked the current evoked by GABA (20 microM); d-tubocurarine (100 microM) only slightly decreased the response to GABA (20 microM). The antagonists were effective over a wide range of holding potentials (-90 mV to +30 mV). The responses to a steady application of both GABA and glycine decayed in a few seconds when recorded under conditions of both symmetric and

  13. Walk the line: 600000 years of molar evolution constrained by allometry in the fossil rodent Mimomys savini

    PubMed Central

    Firmat, Cyril; Lozano-Fernández, Iván; Agustí, Jordi; Bolstad, Geir H.; Cuenca-Bescós, Gloria; Hansen, Thomas F.; Pélabon, Christophe

    2014-01-01

    The allometric-constraint hypothesis states that evolutionary divergence of morphological traits is restricted by integrated growth regulation. In this study, we test this hypothesis on a time-calibrated and well-documented palaeontological sequence of dental measurements on the Pleistocene arvicoline rodent species Mimomys savini from the Iberian Peninsula. Based on 507 specimens representing nine populations regularly spaced over 600 000 years, we compare static (within-population) and evolutionary (among-population) allometric slopes between the width and the length of the first lower molar. We find that the static allometric slope remains evolutionary stable and predicts the evolutionary allometry quite well. These results support the hypothesis that the macroevolutionary divergence of molar traits is constrained by static allometric relationships. PMID:25002706

  14. Walk the line: 600000 years of molar evolution constrained by allometry in the fossil rodent Mimomys savini.

    PubMed

    Firmat, Cyril; Lozano-Fernández, Iván; Agustí, Jordi; Bolstad, Geir H; Cuenca-Bescós, Gloria; Hansen, Thomas F; Pélabon, Christophe

    2014-08-19

    The allometric-constraint hypothesis states that evolutionary divergence of morphological traits is restricted by integrated growth regulation. In this study, we test this hypothesis on a time-calibrated and well-documented palaeontological sequence of dental measurements on the Pleistocene arvicoline rodent species Mimomys savini from the Iberian Peninsula. Based on 507 specimens representing nine populations regularly spaced over 600 000 years, we compare static (within-population) and evolutionary (among-population) allometric slopes between the width and the length of the first lower molar. We find that the static allometric slope remains evolutionary stable and predicts the evolutionary allometry quite well. These results support the hypothesis that the macroevolutionary divergence of molar traits is constrained by static allometric relationships.

  15. Prediction of thyroid C-cell carcinogenicity after chronic administration of GLP1-R agonists in rodents.

    PubMed

    van den Brink, Willem; Emerenciana, Annette; Bellanti, Francesco; Della Pasqua, Oscar; van der Laan, Jan Willem

    2017-04-01

    Increased incidence of C-cell carcinogenicity has been observed for glucagon-like-protein-1 receptor (GLP-1r) agonists in rodents. It is suggested that the duration of exposure is an indicator of carcinogenic potential in rodents of the different products on the market. Furthermore, the role of GLP-1-related mechanisms in the induction of C-cell carcinogenicity has gained increased attention by regulatory agencies. This study proposes an integrative pharmacokinetic/pharmacodynamic (PKPD) framework to identify explanatory factors and characterize differences in carcinogenic potential of the GLP-1r agonist products. PK models for four products (exenatide QW (once weekly), exenatide BID (twice daily), liraglutide and lixisenatide) were developed using nonlinear mixed effects modelling. Predicted exposure was subsequently linked to GLP-1r stimulation using in vitro GLP-1r potency data. A logistic regression model was then applied to exenatide QW and liraglutide data to assess the relationship between GLP-1r stimulation and thyroid C-cell hyperplasia incidence as pre-neoplastic predictor of a carcinogenic response. The model showed a significant association between predicted GLP-1r stimulation and C-cell hyperplasia after 2years of treatment. The predictive performance of the model was evaluated using lixisenatide, for which hyperplasia data were accurately described during the validation step. The use of a model-based approach provided insight into the relationship between C-cell hyperplasia and GLP-1r stimulation for all four products, which is not possible with traditional data analysis methods. It can be concluded that both pharmacokinetics (exposure) and pharmacodynamics (potency for GLP-1r) factors determine C-cell hyperplasia incidence in rodents. Our work highlights the pharmacological basis for GLP-1r agonist-induced C-cell carcinogenicity. The concept is promising for application to other drug classes.

  16. Effects of teicoplanin on cell number of cultured cell lines

    PubMed Central

    Kashkolinejad-Koohi, Tahere; Saadat, Iraj

    2015-01-01

    Teicoplanin is a glycopeptide antibiotic with a wide variation in human serum half-life. It is also a valuable alternative of vancomycin. There is however no study on its effect on cultured cells. The aim of the present study was to test the effect of teicoplanin on cultured cell lines CHO, Jurkat E6.1 and MCF-7. The cultured cells were exposed to teicoplanin at final concentrations of 0–11000 μg/ml for 24 hours. To determine cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. At low concentrations of teicoplanin the numbers of cultured cells (due to cell proliferation) were increased in the three cell lines examined. The maximum cell proliferation rates were observed at concentrations of 1000, 400, and 200 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. Cell toxicity was observed at final concentrations over 2000, 6000, and 400 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. A dose-dependent manner of cell toxicity was observed. Our present findings indicated that teicoplanin at clinically used concentrations induced cell proliferation. It should therefore be used cautiously, particularly in children, pregnant women and patients with cancer. PMID:27486356

  17. Pluripotency of embryo-derived stem cells from rodents, lagomorphs, and primates: Slippery slope, terrace and cliff.

    PubMed

    Savatier, Pierre; Osteil, Pierre; Tam, Patrick P L

    2017-03-01

    The diverse cell states and in vitro conditions for the derivation and maintenance of the mammalian embryo-derived pluripotent stem cells raise the questions of whether there are multiple states of pluripotency of the stem cells of each species, and if there are innate species-specific variations in the pluripotency state. We will address these questions by taking a snapshot of our knowledge of the properties of the pluripotent stem cells, focusing on the maintenance of pluripotency and inter-conversion of the different types of pluripotent stem cells from rodents, lagomorphs and primates. We conceptualize pluripotent stem cells acquiring a series of cellular states represented as terraces on a slope of descending gradient of pluripotency. We propose that reprogramming pluripotent stem cells from a primed to a naive state is akin to moving upstream over a steep cliff to a higher terrace. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  19. Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal.

    PubMed

    Kubota, Hiroshi; Brinster, Ralph L

    2008-01-01

    Spermatogonial stem cells (SSCs), postnatal male germline stem cells, are the foundation of spermatogenesis, during which an enormous number of spermatozoa is produced daily by the testis throughout life of the male. SSCs are unique among stem cells in the adult body because they are the only cells that undergo self-renewal and transmit genes to subsequent generations. In addition, SSCs provide an excellent and powerful model to study stem cell biology because of the availability of a functional assay that unequivocally identifies the stem cell. Development of an in vitro culture system that allows an unlimited supply of SSCs is a crucial technique to manipulate genes of the SSC to generate valuable transgenic animals, to study the self-renewal mechanism, and to develop new therapeutic strategies for infertility. In this chapter, we describe a detailed protocol for the culture of mouse and rat SSCs. A key factor for successful development of the SSC culture system was identification of in vitro growth factor requirements for the stem cell using a defined serum-free medium. Because transplantation assays using immunodeficient mice demonstrated that extrinsic factors for self-renewal of SSCs appear to be conserved among many mammalian species, culture techniques for SSCs of other species, including farm animals and humans, are likely to be developed in the coming 5-10 years.

  20. Profound morphological and functional changes of rodent Purkinje cells between the first and the second postnatal weeks: a metamorphosis?

    PubMed Central

    Dusart, Isabelle; Flamant, Frederic

    2012-01-01

    Between the first and the second postnatal week, the development of rodent Purkinje cells is characterized by several profound transitions. Purkinje cells acquire their typical dendritic “espalier” tree morphology and form distal spines. During the first postnatal week, they are multi-innervated by climbing fibers and numerous collateral branches sprout from their axons, whereas from the second postnatal week, the regression of climbing fiber multi-innervation begins, and Purkinje cells become innervated by parallel fibers and inhibitory molecular layer interneurons. Furthermore, their periods of developmental cell death and ability to regenerate their axon stop and their axons become myelinated. Thus a Purkinje cell during the first postnatal week looks and functions differently from a Purkinje cell during the second postnatal week. These fundamental changes occur in parallel with a peak of circulating thyroid hormone in the mouse. All these features suggest to some extent an interesting analogy with amphibian metamorphosis. PMID:22514522

  1. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    SciTech Connect

    Felthaus, O.; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  2. Investigating citrullinated proteins in tumour cell lines

    PubMed Central

    2013-01-01

    Background The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB). Methods Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines. Results 2-D WB and mass spectrometry identified citrullinated α-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines. Conclusions The citrullination of these proteins suggests a new mechanism in the tumorigenic process. PMID:24099319

  3. Neurogranin is expressed by principal cells but not interneurons in the rodent and monkey neocortex and hippocampus.

    PubMed

    Singec, Ilyas; Knoth, Rolf; Ditter, Margarethe; Volk, Benedikt; Frotscher, Michael

    2004-11-01

    As a substrate of protein kinase C (PKC), neurogranin (NG) is involved in the regulation of calcium signaling and activity-dependent plasticity. Recently, we have shown that, in the rodent cerebellum, NG is exclusively expressed by gamma-aminobutyric acidergic Golgi cells, whereas, in the monkey cerebellum, brush cells were the only neuronal population expressing NG (Singec et al. [2003] J. Comp. Neurol. 459:278-289). In the present study, we analyzed the neocortical and hippocampal expression patterns of NG in adult mouse (C57Bl/6), rat (Wistar), and monkey (Cercopithecus aetiops). By using immunocytochemistry and nonradioactive in situ hybridization, we demonstrate strong NG expression by principal cells in different neocortical layers and in the hippocampus by granule cells of the dentate gyrus and pyramidal neurons of CA1-CA3. In contrast, double-labeling experiments in rodents revealed that neocortical and hippocampal interneurons expressing glutamate decarboxylase 67 (GAD67) were consistently devoid of NG. In addition, by using antibodies against parvalbumin, calbindin, and calretinin, we could demonstrate the absence of NG in interneurons of monkey frontal cortex and hippocampus. Together these findings corroborate the idea of different calcium signaling pathways in excitatory and inhibitory cells that may contribute to different modes of synaptic plasticity in these neurons.

  4. Spontaneous Cell Competition in Immortalized Mammalian Cell Lines

    PubMed Central

    Penzo-Méndez, Alfredo I.; Chen, Yi-Ju; Li, Jinyang; Witze, Eric S.; Stanger, Ben Z.

    2015-01-01

    Cell competition is a form of cell-cell interaction by which cells compare relative levels of fitness, resulting in the active elimination of less-fit cells, “losers,” by more-fit cells, “winners.” Here, we show that in three routinely-used mammalian cell lines – U2OS, 3T3, and MDCK cells – sub-clones arise stochastically that exhibit context-dependent competitive behavior. Specifically, cell death is elicited when winner and loser sub-clones are cultured together but not alone. Cell competition and elimination in these cell lines is caspase-dependent and requires cell-cell contact but does not require de novo RNA synthesis. Moreover, we show that the phenomenon involves differences in cellular metabolism. Hence, our study demonstrates that cell competition is a common feature of immortalized mammalian cells in vitro and implicates cellular metabolism as a mechanism by which cells sense relative levels of “fitness.” PMID:26200654

  5. Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

    PubMed Central

    2009-01-01

    Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use. PMID:19495915

  6. Functional features of cancer stem cells in melanoma cell lines.

    PubMed

    Zimmerer, Rüdiger M; Korn, Philippe; Demougin, Philippe; Kampmann, Andreas; Kokemüller, Horst; Eckardt, André M; Gellrich, Nils-Claudius; Tavassol, Frank

    2013-08-06

    Recent evidence suggests a subset of cells within a tumor with "stem-like" characteristics. These cells are able to transplant tumors in immunodeficient hosts. Distinct from non-malignant stem cells, cancer stem cells (CSC) show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumor cells, and resistance to chemotherapy or radiation. They are often characterized by elevated expression of stem cell surface markers, in particular CD133, and sets of differentially expressed stem cell-associated genes. CSC are usually rare in clinical specimens and hardly amenable to functional studies and gene expression profiling. In this study, a panel of heterogenous melanoma cell lines was screened for typical CSC features. Nine heterogeneous metastatic melanoma cell lines including D10 and WM115 were studied. Cell lines were phenotyped using flow cytometry and clonogenic assays were performed by limiting dilution analysis on magnetically sorted cells. Spheroidal growth was investigated in pretreated flasks. Gene expression profiles were assessed by using real-time rt-PCR and DNA microarrays. Magnetically sorted tumor cells were subcutaneously injected into the flanks of immunodeficient mice. Comparative immunohistochemistry was performed on xenografts and primary human melanoma sections. D10 cells expressed CD133 with a significantly higher clonogenic capacity as compared to CD133- cells. Na8, D10, and HBL cells formed spheroids on poly-HEMA-coated flasks. D10, Me39, RE, and WM115 cells expressed at least 2 of the 3 regulatory core transcription factors SOX2, NANOG, and OCT4 involved in the maintenance of stemness in mesenchymal stem cells. Gene expression profiling on CD133+ and CD133- D10 cells revealed 68 up- and 47 downregulated genes (+/-1.3 fold). Two genes, MGP and PROM1 (CD133), were outstandingly upregulated. CD133+ D10 cells formed tumors in NSG mice contrary to CD133- cells and CD133 expression was detected

  7. Isolation, characterization, and expansion methods for defined primary renal cell populations from rodent, canine, and human normal and diseased kidneys.

    PubMed

    Presnell, Sharon C; Bruce, Andrew T; Wallace, Shay M; Choudhury, Sumana; Genheimer, Christopher W; Cox, Bryan; Guthrie, Kelly; Werdin, Eric S; Tatsumi-Ficht, Patricia; Ilagan, Roger M; Kelley, Russell W; Rivera, Elias A; Ludlow, John W; Wagner, Belinda J; Jayo, Manuel J; Bertram, Timothy A

    2011-03-01

    Chronic kidney disease (CKD) is a global health problem; the growing gap between the number of patients awaiting transplant and organs actually transplanted highlights the need for new treatments to restore renal function. Regenerative medicine is a promising approach from which treatments for organ-level disorders (e.g., neurogenic bladder) have emerged and translated to clinics. Regenerative templates, composed of biodegradable material and autologous cells, isolated and expanded ex vivo, stimulate native-like organ tissue regeneration after implantation. A critical step for extending this strategy from bladder to kidney is the ability to isolate, characterize, and expand functional renal cells with therapeutic potential from diseased tissue. In this study, we developed methods that yield distinct subpopulations of primary kidney cells that are compatible with process development and scale-up. These methods were translated to rodent, large mammal, and human kidneys, and then to rodent and human tissues with advanced CKD. Comparative in vitro studies demonstrated that phenotype and key functional attributes were retained consistently in ex vivo cultures regardless of species or disease state, suggesting that autologous sourcing of cells that contribute to in situ kidney regeneration after injury is feasible, even with biopsies from patients with advanced CKD.

  8. Heme dampens T-cell sequestration by modulating glial cell responses during rodent cerebral malaria.

    PubMed

    Dalko, Esther; Genete, Delphine; Auger, Florent; Dovergne, Claire; Lambert, Claire; Herbert, Fabien; Cazenave, Pierre-André; Roland, Jacques; Pied, Sylviane

    2016-11-01

    Cerebral malaria is the deadliest complication of Plasmodium falciparum infection. Its pathophysiology is associated with a strong pro-inflammatory reaction and the activation of glial cells. Among modulators released during the infection, heme seems to play a controversial role in the pathophysiology of malaria. Herein, we first investigated the phenotype of glial cells during cerebral malaria in C57BL/6 mice infected with P. berghei ANKA. Given the fact that high levels of heme were associated with cerebral malaria, we then investigated its impact on microglial, astrocyte, and T cell responses to further clarify its contribution in the neuropathophysiology. Surprisingly, we found that administration of heme twice a day from day three of infection induced the expression of the Heme oxygenase-1 (Hmox1) gene and prevented brain damages. More specifically, heme inhibited the M1 phenotype of microglia, hampered the activation of astrocytes, and decreased the cerebral expression of Ifng, Tnfa and Ip10. Heme might that way alter the migration of pathogenic CD4 and CD8 T lymphocytes within the brain observed during cerebral malaria. Taking into account that cerebral malaria results from a complex interplay between host- and parasite-derived factors, it is possible that genetic polymorphisms of Hmox1, which could be associated with the control of systemic levels of heme during P. falciparum infection, might explain its dual role and its contribution to the resistance to cerebral malaria. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Glycoprotein 130 receptor signaling mediates α-cell dysfunction in a rodent model of type 2 diabetes.

    PubMed

    Chow, Samuel Z; Speck, Madeleine; Yoganathan, Piriya; Nackiewicz, Dominika; Hansen, Ann Maria; Ladefoged, Mette; Rabe, Björn; Rose-John, Stefan; Voshol, Peter J; Lynn, Francis C; Herrera, Pedro L; Müller, Werner; Ellingsgaard, Helga; Ehses, Jan A

    2014-09-01

    Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines require the glycoprotein 130 (gp130) receptor to signal. In this study, we elucidated the effects of α-cell gp130 receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation increased STAT3 phosphorylation in primary α-cells and stimulated glucagon secretion. Pancreatic α-cell gp130 knockout (αgp130KO) mice showed no differences in glycemic control, α-cell function, or α-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, αgp130KO mice had reduced fasting glycemia, improved glucose tolerance, reduced fasting insulin, and improved α-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of α-cell gp130 receptor signaling has deleterious effects on α-cell function, promoting hyperglycemia. Antagonism of α-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  10. Assignment of adenosine deaminase complexing protein (ADCP) gene(s) to human chromosome 2 in rodent-human somatic cell hybrids.

    PubMed

    Herbschleb-Voogt, E; Grzeschik, K H; Pearson, P L; Meera Khan, P

    1981-01-01

    The experiments reported in this paper indicate that the expression of human adenosine deaminase complexing protein (ADCP) in the human-rodent somatic cell hybrids is influenced by the state of confluency of the cells and the background rodent genome. Thus, the complement of the L-cell derived A9 or B82 mouse parent apparently prevents the expression of human ADCP in the interspecific somatic cell hybrids. In the a3, E36, or RAG hybrids the human ADCP expression was not prevented by the rodent genome and was found to be proportional to the degree of confluency of the cell in the culture as in the case of primary human fibroblasts. An analysis of human chromosomes, chromosome specific enzyme markers, and ADCP in a panel of rodent-human somatic cell hybrids optimally maintained and harvested at full confluency has shown that the expression of human ADCP in the mouse (RAG)-human as well as in the hamster (E36 or a3)-human hybrids is determined by a gene(s) in human chromosome 2 and that neither chromosome 6 nor any other of the chromosomes of man carry any gene(s) involved in the formation of human ADCP at least in the Chinese hamster-human hybrids. A series of rodent-human hybrid clones exhibiting a mitotic separation of IDH1 and MDH1 indicated that ADCP is most probably situated between corresponding loci in human chromosome 2.

  11. Establishment of mouse leukemia cell lines expressing human CD4/CCR5 using lentiviral vectors.

    PubMed

    Li, Ya-Jing; ZhuGe, Fu-Yan; Zeng, Chang-Chun; He, Jin-Yang; Tan, Ning; Liang, Juan

    2017-04-01

    A low-cost rodent model of HIV infection and which presents high application value is an effective tool to investigate HIV infection and pathogenesis. However, development of such a small animal model has been hampered by the unsuitability of rodent cells for HIV-1 replication given that the retrovirus HIV-1 has high selectivity to its host cell. Our study used the mouse leukemia cell lines L615 and L1210 that were induced by murine leukemia virus and transfected with hCD4/CCR5 loaded-lentiviral vector. Lentiviral vectors containing the genes hCD4/CCR5 under the transcriptional control of cytomegalovirus promoter were designed. Transfection efficiencies of human CD4 and CCR5 in L615 and L1210 cells were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot assay. Results showed that hCD4 and CCR5 proteins were expressed on the cell surface, demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells. Moreover, the sensitivity of human CD4/CCR5 transgenic mouse cells to HIV infection was confirmed by RT-PCR and ELISA. Mouse leukemia cell lines that could express hCD4 and CCR5 were thus established to facilitate normal entry of HIV-1 so that a human CD4/CCR5 transgenic mice cell model can be used to investigate the transmission and pathogenesis of HIV/AIDS and potential antiviral drugs against this disease.

  12. Mutagenic processing and strand-specific repair of solar light-induced dipyrimidine photoproducts in rodent cells

    SciTech Connect

    Drobetsky, E.A.; Sage, E.

    1994-12-31

    Although mutations induced by {open_quotes}UVC-like{close_quotes} dipyrimidine photoproducts during sunlight exposure have been directly implicated in multistage photocarcinogenesis, the utility of 254nm UV as a paradigm for broad spectrum solar mutagenesis is questionable. To address this issue, the spectrum of mutations induced by simulated sunlight (SSL) at the adrenine phosphoribosyltransferase locus of Chinese hamster ovary cells has been characterized, and aligned with that previously established for 254nm UV in the same gene. Virtually all SSL-induced events were recovered at potential dinucleotide target sites, most of which could also be implicated in UVC mutagenesis, confirming a preeminent role for cyclobutane dimers and/or (6-4) photoproducts in sunlight-exposed rodent cells. However, striking differences were also noted in the frequency and distribution of mutational classes generated by SSL vs. UVc. Whereas C->T transitions were clearly the most frequent UVC-induced event (58%), three types of base substitution contributed substantially to the SSL spectrum; C->T transitions (35%), T->G transversions (25%), and tandem double CC->TT events (25%). In addition, a significantly greater fraction of SSL-induced mutations (94%) could be attributed to dipyrimidine photoproducts on the non-transcribed strand of the aprt gene than was observed following treatment with 254nm UV (63%). Taken together, the data indicate remarkable differences between UVC- and sunlight-exposed rodent cells in the distribution, strand-specific repair, and/or metabolic processing of similar premutagenic (dipyrimidine) photoproducts.

  13. Fibroblasts From Long-Lived Rodent Species Exclude Cadmium

    PubMed Central

    Dostál, Lubomír; Kohler, William M.; Penner-Hahn, James E.; Miller, Richard A.

    2015-01-01

    Resistance to the lethal effects of cellular stressors, including the toxic heavy metal cadmium (Cd), is characteristic of fibroblast cell lines derived from long-lived bird and rodent species, as well as cell lines from several varieties of long-lived mutant mice. To explore the mechanism of resistance to Cd, we used inductively coupled plasma mass spectroscopy to measure the rate of Cd uptake into primary fibroblasts of 15 rodent species. These data indicate that fibroblasts from long-lived rodent species have slower rates of Cd uptake from the extracellular medium than those from short-lived species. In addition, fibroblasts from short-lived species export more zinc after exposure to extracellular Cd than cells from long-lived species. Lastly, fibroblasts from long-lived rodent species have lower baseline concentrations of two redox-active metals, iron and copper. Our results suggest that evolution of longevity among rodents required adjustment of cellular properties to alter metal homeostasis and to reduce the toxic effects of heavy metals that accumulate over the course of a longer life span. PMID:24522391

  14. Fibroblasts from long-lived rodent species exclude cadmium.

    PubMed

    Dostál, Lubomír; Kohler, William M; Penner-Hahn, James E; Miller, Richard A; Fierke, Carol A

    2015-01-01

    Resistance to the lethal effects of cellular stressors, including the toxic heavy metal cadmium (Cd), is characteristic of fibroblast cell lines derived from long-lived bird and rodent species, as well as cell lines from several varieties of long-lived mutant mice. To explore the mechanism of resistance to Cd, we used inductively coupled plasma mass spectroscopy to measure the rate of Cd uptake into primary fibroblasts of 15 rodent species. These data indicate that fibroblasts from long-lived rodent species have slower rates of Cd uptake from the extracellular medium than those from short-lived species. In addition, fibroblasts from short-lived species export more zinc after exposure to extracellular Cd than cells from long-lived species. Lastly, fibroblasts from long-lived rodent species have lower baseline concentrations of two redox-active metals, iron and copper. Our results suggest that evolution of longevity among rodents required adjustment of cellular properties to alter metal homeostasis and to reduce the toxic effects of heavy metals that accumulate over the course of a longer life span.

  15. SNARE-Mediated Cholesterol Movement to Mitochondria Supports Steroidogenesis in Rodent Cells

    PubMed Central

    Lin, Ye; Hou, Xiaoming; Shen, Wen-Jun; Hanssen, Ruth; Khor, Victor K.; Cortez, Yuan; Roseman, Ann N.; Azhar, Salman

    2016-01-01

    Vesicular transport involving soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins is known to be responsible for many major cellular activities. In steroidogenic tissues, chronic hormone stimulation results in increased expression of proteins involved in the steroidogenic pathway, whereas acute hormone stimulation prompts the rapid transfer of cholesterol to the inner mitochondrial membrane to be utilized as substrate for steroid hormone production. Several different pathways are involved in supplying cholesterol to mitochondria, but mobilization of stored cholesteryl esters appears to initially constitute the preferred source; however, the mechanisms mediating this cholesterol transfer are not fully understood. To study the potential contribution of SNARE proteins in steroidogenesis, we examined the expression levels of various SNARE proteins in response to hormone stimulation in steroidogenic tissues and cells and established an in vitro mitochondria reconstitution assay system to assess the contribution of various SNARE proteins on cholesterol delivery for steroidogenesis. Our results from reconstitution experiments along with knockdown studies in rat primary granulosa cells and in a Leydig cell line show that soluble N-ethylmaleimide sensitive factor attachment protein-α, synaptosomal-associated protein of 25 kDa, syntaxin-5, and syntaxin-17 facilitate the transport of cholesterol to mitochondria. Thus, although StAR is required for efficient cholesterol movement into mitochondria for steroidogenesis, specific SNAREs participate and are necessary to mediate cholesterol movement to mitochondria. PMID:26771535

  16. Pancreastatin producing cell line from human pancreatic islet cell tumor.

    PubMed

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Ikeda, Y; Kono, A

    1990-04-30

    It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponentially growing cultures produced 5.7 fmol of pancreastatin/10(6) cells/hr. Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the tumor. Gel filtrations of culture medium and tumor extract identified heterogenous molecular forms of PST-LI which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin.

  17. GPVI oligomerisation in cell lines and platelets

    PubMed Central

    2007-01-01

    Summary Background Glycoprotein VI (GPVI) is a physiological receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerisation of GPVI has been proposed as necessary for the interaction with collagen, although direct evidence of dimerisation has not been reported in cell lines or platelets. Objectives To investigate oligomerisation of GPVI in transfected cell lines and in platelets under nonstimulated conditions. Methods and Results By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extra-cellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerisation of GPVI under these conditions. A chemical cross-linker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the FcR γ-chain. Conclusions The present results directly demonstrate GPVI-FcR γ-chain oligomerisation at the surface of the platelet, and thereby add to the growing evidence that oligomerisation of GPVI may be a pre-requisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage. PMID:17367493

  18. Effect of glutamate analogues on brain tumor cell lines.

    PubMed

    Campbell, G L; Bartel, R; Freidman, H S; Bigner, D D

    1985-10-01

    Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.

  19. High prevalence of side population in human cancer cell lines

    PubMed Central

    Boesch, Maximilian; Zeimet, Alain G.; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. PMID:27226981

  20. A bioinformatics analysis of the cell line nomenclature.

    PubMed

    Sarntivijai, Sirarat; Ade, Alexander S; Athey, Brian D; States, David J

    2008-12-01

    Cell lines are used extensively in biomedical research, but the nomenclature describing cell lines has not been standardized. The problems are both linguistic and experimental. Many ambiguous cell line names appear in the published literature. Users of the same cell line may refer to it in different ways, and cell lines may mutate or become contaminated without the knowledge of the user. As a first step towards rationalizing this nomenclature, we created a cell line knowledgebase (CLKB) with a well-structured collection of names and descriptive data for cell lines cultured in vitro. The objectives of this work are: (i) to assist users in extracting useful information from biomedical text and (ii) to highlight the importance of standardizing cell line names in biomedical research. This CLKB contains a broad collection of cell line names compiled from ATCC, Hyper CLDB and MeSH. In addition to names, the knowledgebase specifies relationships between cell lines. We analyze the use of cell line names in biomedical text. Issues include ambiguous names, polymorphisms in the use of names and the fact that some cell line names are also common English words. Linguistic patterns associated with the occurrence of cell line names are analyzed. Applying these patterns to find additional cell line names in the literature identifies only a small number of additional names. Annotation of microarray gene expression studies is used as a test case. The CLKB facilitates data exploration and comparison of different cell lines in support of clinical and experimental research. The web ontology file for this cell line collection can be downloaded at http://www.stateslab.org/data/celllineOntology/cellline.zip.

  1. Transplantation of human neural stem cells restores cognition in an immunodeficient rodent model of traumatic brain injury.

    PubMed

    Haus, Daniel L; López-Velázquez, Luci; Gold, Eric M; Cunningham, Kelly M; Perez, Harvey; Anderson, Aileen J; Cummings, Brian J

    2016-07-01

    Traumatic brain injury (TBI) in humans can result in permanent tissue damage and has been linked to cognitive impairment that lasts years beyond the initial insult. Clinically effective treatment strategies have yet to be developed. Transplantation of human neural stem cells (hNSCs) has the potential to restore cognition lost due to injury, however, the vast majority of rodent TBI/hNSC studies to date have evaluated cognition only at early time points, typically <1month post-injury and cell transplantation. Additionally, human cell engraftment and long-term survival in rodent models of TBI has been difficult to achieve due to host immunorejection of the transplanted human cells, which confounds conclusions pertaining to transplant-mediated behavioral improvement. To overcome these shortfalls, we have developed a novel TBI xenotransplantation model that utilizes immunodeficient athymic nude (ATN) rats as the host recipient for the post-TBI transplantation of human embryonic stem cell (hESC) derived NSCs and have evaluated cognition in these animals at long-term (≥2months) time points post-injury. We report that immunodeficient ATN rats demonstrate hippocampal-dependent spatial memory deficits (Novel Place, Morris Water Maze), but not non-spatial (Novel Object) or emotional/anxiety-related (Elevated Plus Maze, Conditioned Taste Aversion) deficits, at 2-3months post-TBI, confirming that ATN rats recapitulate some of the cognitive deficits found in immunosufficient animal strains. Approximately 9-25% of transplanted hNSCs survived for at least 5months post-transplantation and differentiated into mature neurons (NeuN, 18-38%), astrocytes (GFAP, 13-16%), and oligodendrocytes (Olig2, 11-13%). Furthermore, while this model of TBI (cortical impact) targets primarily cortex and the underlying hippocampus and generates a large lesion cavity, hNSC transplantation facilitated cognitive recovery without affecting either lesion volume or total spared cortical or hippocampal

  2. On the Ontology Based Representation of Cell Lines

    PubMed Central

    Ganzinger, Matthias; He, Shan; Breuhahn, Kai; Knaup, Petra

    2012-01-01

    Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT) integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed. PMID:23144907

  3. Toward a More Precise and Informative Nomenclature Describing Fetal and Neonatal Male Germ Cells in Rodents1

    PubMed Central

    McCarrey, John R.

    2013-01-01

    ABSTRACT The germ cell lineages are among the best characterized of all cell lineages in mammals. This characterization includes precise nomenclature that distinguishes among numerous, often subtle, changes in function or morphology as development and differentiation of germ cells proceed to form the gametes. In male rodents, there are at least 41 distinct cell types that occur during progression through the male germ cell lineage that gives rise to spermatozoa. However, there is one period during male germ cell development—that which occurs immediately following the primordial germ cell stage and prior to the spermatogonial stage—for which the system of precise and informative cell type terminology is not adequate. Often, male germ cells during this period are referred to simply as “gonocytes.” However, this term is inadequate for multiple reasons, and it is suggested here that nomenclature originally proposed in the 1970s by Hilscher et al., which employs the terms M-, T1-, and T2-prospermatogonia, is preferable. In this Minireview, the history, proper utilization, and advantages of this terminology relative to that of the term gonocytes are described. PMID:23843236

  4. EXAFS studies of prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Czapla, J.; Kwiatek, W. M.; Lekki, J.; Kisiel, A.; Steininger, R.; Goettlicher, J.

    2013-04-01

    Sulphur plays a vital role in every human organism. It is known, that sulphur-bearing compounds, such as for example cysteine and glutathione, play critical roles in development and progression of many diseases. Any alteration in sulphur's biochemistry could become a precursor of serious pathological conditions. One of such condition is prostate cancer, the most frequently diagnosed malignancy in the western world and the second leading cause of cancer related death in men. The purpose of presented studies was to examine what changes occur in the nearest chemical environment of sulphur in prostate cancer cell lines in comparison to healthy cells. The Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used, followed by theoretical calculations. The results of preliminary analysis is presented.

  5. 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

  6. Satellite cell activation induced by aerobic muscle adaptation in response to endurance exercise in humans and rodents.

    PubMed

    Abreu, Phablo; Mendes, Sávio Victor Diógenes; Ceccatto, Vânia Marilande; Hirabara, Sandro Massao

    2017-02-01

    Although the requirement of satellite cells activation and expansion following injury, mechanical load or growth stimulus provoked by resistance exercise has been well established, their function in response to aerobic exercise adaptation remains unclear. A clear relationship between satellite cell expansion in fiber-type specific myosin heavy chain and aerobic performance has been related, independent of myonuclear accretion or muscle growth. However, the trigger for this activation process is not fully understood yet and it seems to be a multi-faceted and well-orchestrated process. Emerging in vitro studies suggest a role for metabolic pathways and oxygen availability for satellite cell activation, modulating the self-renewal potential and cell fate control. The goal of this review is to describe and discuss the current knowledge about the satellite cell activation and expansion in response to aerobic exercise adaptation in human and rodent models. Additionally, findings about the in vitro metabolic control, which seems be involved in the satellite cell activation and cell fate control, are presented and discussed.

  7. Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines

    PubMed Central

    Fernández-Moreno, Mercedes; Hermida-Gómez, Tamara; Gallardo, M. Esther; Dalmao-Fernández, Andrea; Rego-Pérez, Ignacio; Garesse, Rafael

    2016-01-01

    Introduction The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. Methodology Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. Results The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic

  8. Detection of rodent coronaviruses in tissues and cell cultures by using polymerase chain reaction.

    PubMed Central

    Homberger, F R; Smith, A L; Barthold, S W

    1991-01-01

    A polymerase chain reaction (PCR) method was developed for the detection of rodent coronaviruses in biological material by using reverse transcriptase and two primers which flanked an M gene sequence of 375 bp. PCR detected all of 11 different strains of mouse hepatitis virus (MHV) as well as rat sialodacryoadenitis virus but not bovine coronavirus or human coronavirus strains OC43 and 229E. The M gene sequences of bovine coronavirus and human coronavirus OC43 are homologous to that of MHV, but minor differences exist in the primer regions, preventing annealing of the primers. For detecting MHV-Y in tissue samples, PCR was faster than and at least as sensitive as either of the two bioassays (infant mouse bioassay and mouse antibody production test) currently used for MHV diagnostic purposes. Images PMID:1661745

  9. A Neuroblastoma × Glioma Hybrid Cell Line with Morphine Receptors

    PubMed Central

    Klee, Werner A.; Nirenberg, Marshall

    1974-01-01

    A neuroblastoma × glioma hybrid cell line with well-developed neural properties was found that has high-affinity morphine receptors. The average cell contains approximately 3 × 106 receptors. In contrast, parent cells and other neuroblastoma or hybrid cell lines tested had few or no morphine receptors. PMID:4530316

  10. A rodent model of metabolic surgery for study of type 2 diabetes and positron emission tomography scanning of beta cell mass

    PubMed Central

    Inabnet, William B.; Milone, Luca; Korner, Judith; Durak, Evren; Ahmed, Leaque; Pomrantz, Jill; Harris, Paul E.; Bessler, Marc

    2013-01-01

    Background Type 2 diabetes mellitus is a worldwide healthcare problem with major socioeconomic implications. Metabolic surgical procedures have been shown to improve diabetes, but the mechanism of action is poorly understood. The Goto-Kakizaki (GK) rodent is a type 2 diabetic animal model that is ideally situated for studying the effect of surgery on diabetes; however, the operative mortality is high. The aim of this study was to describe the operative technique, improvements in perioperative management, and the technique of micro-positron emission tomography (PET) scanning of the β-cell mass in GK rodents. Methods A total of 53 GK rats were divided into 1 of 3 operative groups: sham, sleeve gastrectomy, and duodenojejunal bypass. A subset of animals underwent micro-PET scanning with [11C]-dihydrotetrabenazine to determine the vesicular monoamine transporter 2 binding index, an indicator of β-cell mass. Results The 30-day mortality in the sham and sleeve gastrectomy rodents was 0; however, 2 sleeve gastrectomy rodents developed enterocutaneous fistula and 1 developed an abscess. In the duodenojejunal bypass group, the initial mortality rate was close to 90%; however, refinements in the surgical technique and perioperative management (fluids, antibiotics, pain control) lowered the mortality rate to 60%. The surgical technique is discussed in detail. [11C]-Dihydrotetrabenazine uptake in the pancreas was demonstrated on micro-PET scanning in the sham and duodenojejunal bypass rodents. Conclusion Intensive medical management in the perioperative period and attention to the operative technique lowered the mortality. [11C]-Dihydrotetrabenazine micro-PET scanning is a feasible method for assessing the β-cell mass in GK rodents and could prove to be an important modality for evaluating β-cell performance in type 2 diabetes. PMID:19136315

  11. DNA profiling and characterization of animal cell lines.

    PubMed

    Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

    2014-01-01

    The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines.

  12. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  13. Tissue expander stimulated lengthening of arteries (TESLA) induces early endothelial cell proliferation in a novel rodent model.

    PubMed

    Potanos, Kristina; Fullington, Nora; Cauley, Ryan; Purcell, Patricia; Zurakowski, David; Fishman, Steven; Vakili, Khashayar; Kim, Heung Bae

    2016-04-01

    We examine the mechanism of aortic lengthening in a novel rodent model of tissue expander stimulated lengthening of arteries (TESLA). A rat model of TESLA was examined with a single stretch stimulus applied at the time of tissue expander insertion with evaluation of the aorta at 2, 4 and 7day time points. Measurements as well as histology and proliferation assays were performed and compared to sham controls. The aortic length was increased at all time points without histologic signs of tissue injury. Nuclear density remained unchanged despite the increase in length suggesting cellular hyperplasia. Cellular proliferation was confirmed in endothelial cell layer by Ki-67 stain. Aortic lengthening may be achieved using TESLA. The increase in aortic length can be achieved without tissue injury and results at least partially from cellular hyperplasia. Further studies are required to define the mechanisms involved in the growth of arteries under increased longitudinal stress. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

    NASA Astrophysics Data System (ADS)

    Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

    2017-05-01

    The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

  15. Evolution of T-Cell Receptor Gamma and Delta Constant Region and Other T-Cell-Related Proteins in the Human-Rodent-Artiodactyl Triplet

    PubMed Central

    Ciccarese, S.; Lanave, C.; Saccone, C.

    1997-01-01

    In this paper we report a detailed comparative and evolutionary analysis of the sequences of constant T-cell receptor (Tcr) Cγδ genes of artiodactyls compared to the homologous sequences of rodents and primates. Because of the frequency and physiological distribution of γδ T-cells in different animals, rodents and humans are defined as ``γδ low'' species and ruminants as ``γδ high'' species. Such a characteristic seems to be due to an adaptive role of γδ T-cell function. By analyzing the ruminant gene phylogeny of Tcr Cγ we were able to estimate the distance between cattle and sheep at 18 million years ago, a time that is in agreement with other nonmolecular estimates. For Tcr Cγδ genes a peculiar phylogenetic relationship was found, with human and mouse clustering together and leaving artiodactyls apart. By using appropriate outgroups, the same phylogenetic pattern was obtained with other T-cell related sequences: namely, Tcr Cα chain, CD3 γ and δ invariant subunits, Interleukin-2, Interleukin-2 receptor α chain and Interleukin-1β with the exception of Tcr Cβ chain and Interleukin-1α. In contrast, the analysis of all other T-cell nonrelated genes available in primary databases reveals a different tree, where primates and artiodactyls are sister taxa and rodents are apart in accordance with the current view of mammalian phylogeny. These data are relevant to important evolutionary issues. They show how misleading a phylogeny based on a single or on a few homologous genes may be. In addition they demonstrate that genes with correlated functions may evolve in a lineage specific manner probably in relation to environmental conditions. PMID:9071594

  16. DNA strand breaks induced in cultured human and rodent cells by chlorohydroxyfuranones--mutagens isolated from drinking water

    SciTech Connect

    Chang, L.W.; Daniel, F.B.; DeAngelo, A.B. )

    1991-01-01

    Chlorohydroxyfuranones, by-products of chlorine disinfection and drinking water contaminants, are shown to produce DNA strand breaks in human and rodent cells. One chlorohydroxyfuranone, 3-chloro-4-dichloromethyl-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen, induces 232 +/- 89 DNA strand breaks.(cell-microM)-1 in human CCRF-CEM cells over a concentration range of 4.4 to 220 microM. This constitutes a DNA damage potency comparable to dimethylsulfate (DMS). By comparison, 3,4-dichloro-5-hydroxy-2(5H)-furanone (MA), another chlorohydroxyfuranone which is approximately four orders of magnitude less mutagenic than MX in Salmonella typhimurium strain TA100, is only about tenfold less potent as an inducer of DNA strand breaks in these cells, i.e., 18.2 +/- 3.1 strand breaks.(cell-microM)-1. The DNA strand-breaking potential of MX is inactivated by prior incubation with a rat liver S9 homogenate. In addition, both chlorohydroxyfuranones are ineffective at producing DNA strand breaks in primary rate hepatocytes (PRH) at concentrations below those which produce cytotoxicity as assessed by release of the cellular enzyme lactate dehydrogenase (LDH). Prior treatment of the PRH with 750 microM diethyl maleate, a glutathione-depleting agent, did not enhance the cytotoxicity nor the DNA strand-breaking potential of either chlorohydroxyfuranone. This could indicate that glutathione-glutathione-S-transferase is not an important mechanism for the detoxification of these compounds in PRH.

  17. Host cell/Orientia tsutsugamushi interactions: evolution and expression of syndecan-4 in Asian rodents (Rodentia, Muridae).

    PubMed

    Badenhorst, Daleen; Tatard, Caroline; Suputtamongkol, Yupin; Robinson, Terence J; Dobigny, Gauthier

    2012-07-01

    Scrub typhus is an acute febrile zoonotic disease and worldwide more than a billion people may be at risk for infection. Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium. Rodents are reported to be the primary reservoir hosts of the disease and according to the most recent surveys, all species within the Rattus sensu lato complex of the tribe Rattini are carriers of scrub typhus. There is no evidence that any of mouse (Mus) species serves as the primary reservoir of the bacterium even when occurring in sympatry with wild infected rats. This contrast in the host/syndecan-4 interactions between Rattini and Asian Murini may be due to intrinsic (i.e., genetic) differences. Herein we compare the sequence and expression levels of syndecan-4 (the putative cell receptor of O. tsutsugamushi) between Rattini species that are known to be natural reservoirs for the typhus agents, and Murini species that are not. Although it was not possible to conclusively link the structural variations detected in syndecan-4 with carrier status in either Rattini and Murini, our findings indicate the absence of a strong Orientia-mediated selective regime acting on gene structure. In contrast, variable spleen-specific syndecan-4 expression levels show a strong correlation between under-expression of syndecan-4 in Murini and seropositive Rattini, compared to seronegative Rattini rodents. We postulate that two divergent responses may be at work in Murini and Rattini, both linked with differential expression of syndecan-4: (i) reduced syndecan-4 transcription in Murini decreases the likelihood that the host cells will become infected by the Orientia bacterium, while (ii) reduced syndecan-4 expression in seropositive Rattini limits the pathogenicity of Orientia and consequently improves the longevity of the rat hosts. These patterns may underpin the poor carrier status of wild mice on the one hand, and the effective role of wild rats as reservoir

  18. Rodent Anterior Ischemic Optic Neuropathy (rAION) Induces Regional Retinal Ganglion Cell Apoptosis with a Unique Temporal Pattern

    PubMed Central

    Slater, Bernard J.; Mehrabian, Zara; Guo, Yan; Hunter, Allan

    2009-01-01

    Purpose Nonarteritic anterior ischemic optic neuropathy (NAION) results in optic nerve damage with retinal ganglion cell (RGC) loss. An NAION model, rodent anterior ischemic optic neuropathy (rAION), was used to determine AION-associated mechanisms of RGC death and associated regional retinal changes. Methods rAION was induced in male Wistar rats, and the retinas analyzed at various times after induction. RGCs were positively identified by both retrograde fluorogold labeling and brain-expressed X-linked protein-1/2 (Bex1/2) immunoreactivity. RGC death was analyzed by fluorescein-tagged annexin-V labeling (FITC-annexin-V), as well as by terminal nucleotide nick-end labeling (TUNEL). Retinal flatmount preparations enabled regional retinal analysis of labeled dying cells. Apoptosis pathway activation was confirmed by Western analysis, with an antibody that recognizes cleaved caspase-3. Results Post-rAION, RGCs die by apoptosis over a longer period than previously recognized. Cleaved caspase-3 immunoreactivity was greatest between 11 and 15 days. rAION-induced RGC death occurs regionally, with sparing of large contiguous regions of RGCs. Conclusions rAION results in later RGC death than in traumatic optic nerve damage models. Apoptosis, measured by FITC-annexin, occurs maximally in the second to third week after infarct. Cleaved caspase-3 activation confirms that after rAION, RGCs undergo apoptosis by the caspase activation pathway. The regional pattern in dying RGCs after rAION implies that a measure of retinotopic organization occurs in the rodent optic nerve. The prolonged period from insult to death suggests that the window for successful treatment after ON infarct may be longer than previously recognized. PMID:18660428

  19. Rodent repellency

    USGS Publications Warehouse

    DeWitt, J.B.; Welch, J.F.; Bellack, E.

    1950-01-01

    In the course of studies involving more than 2,500 chemical repellents, it has been found that certain groups of- compounds containing nitrogen or sulfur are repellent to rats under the , test conditions and it appears probable that some of these compounds might be used for the protection of packaged goods against rodent attacks. Additional tests to determine optimum methods of application will be necessary before final evaluation of these compounds will be possible and extensive field trials will be required to establish the degree of protection which may be afforded by the use of these materials. Pending such final evaluation, it may be assumed that the results,to date offer a means of selecting the most promising types of'materials for further trial....On the basis of the test data, it appears that some amine derivative, such as a salt of some organic, acid, or a complex with trinitrobenzene or with a metallic salt of a dialkyl dithiocarbamic acid might offer promise of protection of packaging materials against rodent attacks....Protection might be obtained through the use of certain 'physical deterrents' such as plastics, waxes or drying oils.

  20. Sensitivities of NIH/3T3-derived clonal cell lines to ionizing radiation: Significance for gene transfer studies

    SciTech Connect

    Kasid, U.N.; Weichselbaum, R.R.; Brennan, T.; Mark, G.E.; Dritschilo, A. )

    1989-06-15

    Rodent cells are frequently used as recipients in experiments involving gene transfer, isolation, and characterization. The present studies were designed to investigate the clonal responses to ionizing radiation of NIH/3T3 cells subjected to DNA-mediated gene transfer. Radiation sensitivity (D0) values were determined for the parental NIH/3T3 cell strain, six clonal cell lines transfected with DNA from radiation-resistant human tumor cells, and six nontransfected clonal cell lines. The radiation sensitivities of four transfected and two nontransfected clonal cell lines differed significantly from parental NIH/3T3 cells (P less than 0.05). Detailed karyotype analysis of two nontransfected clonal cell lines with differing radiation sensitivities showed variation in chromosomal composition. Specifically, a minute chromosome was observed to segregate consistently (in 49 of 50 metaphases) with the genome of one NIH/3T3 clone (D0 2.07 Gy) and was completely absent (from 50 metaphases) in another NIH/3T3 clone (D0 1.06 Gy). In the parental NIH/3T3 strain (D0 2.02 Gy) 10% of cells (3 of 30 metaphases) had such minute chromosomes. These findings demonstrate that the clonal cellular heterogeneity of NIH/3T3 cells is characterized by genotypic and phenotypic variations which must be considered in the experimental design involving gene transfer and expression.

  1. A cell kinetic model of granulopoiesis under radiation exposure: extension from rodents to canines and humans.

    PubMed

    Hu, Shaowen; Cucinotta, Francis A

    2011-02-01

    As significant ionising radiation exposure will occur during prolonged space travel in future, it is essential to understand their adverse effects on the radiosensitive organ systems that are important for immediate survival of humans, e.g. the haematopoietic system. In this paper, a biomathematical model of granulopoiesis is used to analyse the granulocyte changes seen in the blood of mammalians under acute and continuous radiation exposure. This is one of a set of haematopoietic models that have been successfully utilised to simulate and interpret the experimental data of acute and chronic radiation on rodents. Extension to canine and human systems indicates that the results of the model are consistent with the cumulative experimental and empirical data from various sources, implying the potential to integrate them into one united model system to monitor the haematopoietic response of various species under irradiation. The suppression of granulocytes' level of a space traveller under chronic stress of low-dose irradiation as well as the granulopoietic response when encountering a historically large solar particle event is also discussed.

  2. Cell-Type and State-Dependent Synchronization among Rodent Somatosensory, Visual, Perirhinal Cortex, and Hippocampus CA1.

    PubMed

    Vinck, Martin; Bos, Jeroen J; Van Mourik-Donga, Laura A; Oplaat, Krista T; Klein, Gerbrand A; Jackson, Jadin C; Gentet, Luc J; Pennartz, Cyriel M A

    2015-01-01

    Beta and gamma rhythms have been hypothesized to be involved in global and local coordination of neuronal activity, respectively. Here, we investigated how cells in rodent area S1BF are entrained by rhythmic fluctuations at various frequencies within the local area and in connected areas, and how this depends on behavioral state and cell type. We performed simultaneous extracellular field and unit recordings in four connected areas of the freely moving rat (S1BF, V1M, perirhinal cortex, CA1). S1BF spiking activity was strongly entrained by both beta and gamma S1BF oscillations, which were associated with deactivations and activations, respectively. We identified multiple classes of fast spiking and excitatory cells in S1BF, which showed prominent differences in rhythmic entrainment and in the extent to which phase locking was modulated by behavioral state. Using an additional dataset acquired by whole-cell recordings in head-fixed mice, these cell classes could be compared with identified phenotypes showing gamma rhythmicity in their membrane potential. We next examined how S1BF cells were entrained by rhythmic fluctuations in connected brain areas. Gamma-synchronization was detected in all four areas, however we did not detect significant gamma coherence among these areas. Instead, we only found long-range coherence in the theta-beta range among these areas. In contrast to local S1BF synchronization, we found long-range S1BF-spike to CA1-LFP synchronization to be homogeneous across inhibitory and excitatory cell types. These findings suggest distinct, cell-type contributions of low and high-frequency synchronization to intra- and inter-areal neuronal interactions.

  3. Cell-Type and State-Dependent Synchronization among Rodent Somatosensory, Visual, Perirhinal Cortex, and Hippocampus CA1

    PubMed Central

    Vinck, Martin; Bos, Jeroen J.; Van Mourik-Donga, Laura A.; Oplaat, Krista T.; Klein, Gerbrand A.; Jackson, Jadin C.; Gentet, Luc J.; Pennartz, Cyriel M. A.

    2016-01-01

    Beta and gamma rhythms have been hypothesized to be involved in global and local coordination of neuronal activity, respectively. Here, we investigated how cells in rodent area S1BF are entrained by rhythmic fluctuations at various frequencies within the local area and in connected areas, and how this depends on behavioral state and cell type. We performed simultaneous extracellular field and unit recordings in four connected areas of the freely moving rat (S1BF, V1M, perirhinal cortex, CA1). S1BF spiking activity was strongly entrained by both beta and gamma S1BF oscillations, which were associated with deactivations and activations, respectively. We identified multiple classes of fast spiking and excitatory cells in S1BF, which showed prominent differences in rhythmic entrainment and in the extent to which phase locking was modulated by behavioral state. Using an additional dataset acquired by whole-cell recordings in head-fixed mice, these cell classes could be compared with identified phenotypes showing gamma rhythmicity in their membrane potential. We next examined how S1BF cells were entrained by rhythmic fluctuations in connected brain areas. Gamma-synchronization was detected in all four areas, however we did not detect significant gamma coherence among these areas. Instead, we only found long-range coherence in the theta-beta range among these areas. In contrast to local S1BF synchronization, we found long-range S1BF-spike to CA1–LFP synchronization to be homogeneous across inhibitory and excitatory cell types. These findings suggest distinct, cell-type contributions of low and high-frequency synchronization to intra- and inter-areal neuronal interactions. PMID:26834582

  4. Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood.

    PubMed

    Rådinger, Madeleine; Jensen, Bettina M; Kuehn, Hye Sun; Kirshenbaum, Arnold; Gilfillan, Alasdair M

    2010-08-01

    Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identify potential ways to prevent them, have primarily been conducted in rodent mast cells. However, to understand how these responses pertain to human disease, and to investigate and develop novel therapies for the treatment of human mast cell-driven disease, human mast cell models may have greater relevance. Recently, a number of systems have been developed to allow investigators to readily obtain sufficient quantities of human mast cells to conduct these studies. These mast cells release the appropriate suite of inflammatory mediators in response to known mast cell activators including antigen. These systems have also been employed to examine the signaling events regulating these responses. Proof of principle studies has also demonstrated utility of these systems for the identification of potential inhibitors of mast cell activation and growth. In this unit, techniques for the development and culture of human mast cells from their progenitors and the culture of human mast cell lines are described. The relative merits and drawbacks of each model are also described.

  5. Immunoglobulin expression and synthesis by human haemic cell lines.

    PubMed Central

    Gordon, J; Hough, D; Karpas, A; Smith, J L

    1977-01-01

    Twenty-six human cell lines derived from a variety of lymphoid and non-lymphoid malignancies, were investigated for their immunological markers, with special reference to the class of immunoglobulin expressed. Twenty-five of the lines stained positively for surface immunoglobulin and IgD together with IgM proved to be the major immunoglobulin classes on these cells. Six of the lines were chosen for a study of their immunoglobulin synthesis patterns over an 18-h period and the immunoglobulin produced was analysed on SDS-polyacrylamide gel electrophoresis. Patterns obtained from the cell lines were similar to that from normal lymph node lymphocytes and differed markedly to plasma cells. Two of the cell lines had abnormal immunoglobulin synthesis patterns characterized as free light chains in one case. The cell lines are evaluated for their usefulness as models of immunoglobulin synthesis and analogues of normal and neoplastic states. PMID:608682

  6. Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

    PubMed

    Kerr, M; Fischer, J E; Purushotham, K R; Gao, D; Nakagawa, Y; Maeda, N; Ghanta, V; Hiramoto, R; Chegini, N; Humphreys-Beher, M G

    1994-08-02

    The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was

  7. Serum-free bioprocessing of adult human and rodent skin-derived Schwann cells: implications for cell therapy in nervous system injury.

    PubMed

    Mirfeizi, Leila; Stratton, Jo Anne; Kumar, Ranjan; Shah, Prajay; Agabalyan, Natacha; Stykel, Morgan G; Midha, Rajiv; Biernaskie, Jeff; Kallos, Michael S

    2017-02-08

    Peripheral nerve injury affects 2.8% of trauma patients with severe cases often resulting in long-lived permanent disability, despite nerve repair surgery. Autologous Schwann cell (SC) therapy currently provides an exciting avenue for improved outcomes for these patients, particularly with the possibility to derive SCs from easily-accessible adult skin. However, due to current challenges regarding the efficient expansion of these cells, further optimization is required before they can be seriously considered for clinical application. Here, a microcarrier-based bioreactor system is proposed as a means to scale-up large numbers of adult skin-derived SCs for transplantation into the injured nerve. Bioprocessing parameters that allow for the expansion of adult rodent SCs have been identified, whilst maintaining similar rates of proliferation (as compared to static-grown SCs), expression of SC markers, and, importantly, their capacity to myelinate axons following transplant into the injured sciatic nerve. The same bioprocessing parameters can be applied to SCs derived from adult human skin, and like rodent cells, they sustain their proliferative potential and expression of SC markers. Taken together, this dataset demonstrates the basis for a scalable bioprocess for the production of SCs, an important step towards clinical use of these cells as an adjunct therapy for nerve repair. Copyright © 2017 John Wiley & Sons, Ltd.

  8. The transcriptome of the Didelphis virginiana opossum kidney OK proximal tubule cell line.

    PubMed

    Eshbach, Megan L; Sethi, Rahil; Avula, Raghunandan; Lamb, Janette; Hollingshead, Deborah J; Finegold, David N; Locker, Joseph D; Chandran, Uma R; Weisz, Ora A

    2017-09-01

    The OK cell line derived from the kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, the genomic sequence for D. virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The M. domestica sequence is not highly annotated, and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the D. virginiana OK cell line, we characterized its transcriptome via de novo transcriptome assembly and alignment to the M. domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the M. domestica genome, were compared with publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT-specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells. Copyright © 2017 the American Physiological Society.

  9. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R.

    1989-01-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

  10. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

  11. Continuous human cell lines and method of making same

    SciTech Connect

    Stampfer, M.R.

    1989-02-28

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. No Drawings

  12. Susceptibilities of 14 cell lines to bluetongue virus infection.

    PubMed Central

    Wechsler, S J; McHolland, L E

    1988-01-01

    The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations. PMID:2853175

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

    USDA-ARS?s Scientific Manuscript database

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

  14. Interference with protease-activated receptor 1 does not reduce damage to subventricular zone cells of immature rodent brain following exposure to blood or blood plasma.

    PubMed

    Mao, Xiaoyan; Del Bigio, Marc R

    2015-02-04

    Prior work showed that whole blood, plasma, and serum injections are damaging to the neonatal rodent brain in a model of intracerebral/periventricular hemorrhage. Thrombin alone is also damaging. In adult animal models of hemorrhagic stroke, the protease-activated (thrombin) receptor PAR1 mediates some of the brain damage. We hypothesized that PAR1 interference will reduce the adverse effects of blood products on immature rodent brain and cells. Cultured oligodendrocyte precursor cells from rats and mice were exposed to blood plasma with and without the PAR1 antagonists SCH-79797 or BMS-200261. In concentrations previously shown to have activity on brain cells, neither drug showed evidence of protection against the toxicity of blood plasma. Newborn mice (wild type, heterozygous, and PAR1 knockout) were subjected to intracerebral injection of autologous whole blood into the periventricular region of the frontal lobe. Cell proliferation, measured by Ki67 immunoreactivity in the subventricular zone, was suppressed at 1 and 2 days, and was not normalized in the knockout mice. Cell apoptosis, measured by activated caspase 3 immunoreactivity, was not apparent in the subventricular zone. Increased apoptosis in periventricular striatal cells was not normalized in the knockout mice. Interference with the thrombin-PAR1 system does not reduce the adverse effects of blood on germinal cells of the immature rodent brain. PAR1 interference is unlikely to be a useful treatment for reducing the brain damage that accompanies periventricular (germinal matrix) hemorrhage, a common complication of premature birth.

  15. Re-characterization of established human retinoblastoma cell lines.

    PubMed

    Busch, Maike; Philippeit, Claudia; Weise, Andreas; Dünker, Nicole

    2015-03-01

    Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro.

  16. The pursuit of ES cell lines of domesticated ungulates

    USDA-ARS?s Scientific Manuscript database

    In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated state, have the ability to self-renew, and exhibit pluripotency, i.e., they can give rise to most if not all somatic cell types and to the germ cells, egg and sperm. These characteristics make ES cell lines...

  17. Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)

    PubMed Central

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Meng, Tao; Chow, Vincent TK; Chua, Kaw Bing

    2016-01-01

    Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1K98E,E145A,L169F with three substitutions in the VP1 protein—K98E, E145A and L169F—productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection. PMID:27329847

  18. Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain).

    PubMed

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Meng, Tao; Chow, Vincent Tk; Chua, Kaw Bing

    2016-06-22

    Enterovirus 71 (EV71) is a neurotrophic virus that causes hand, foot and mouth disease (HFMD) and occasional neurological infection among children. It infects primate cells but not rodent cells, primarily due to the incompatibility between the virus and the expressed form of its receptor, scavenger receptor class B member 2 (SCARB2) protein, on rodent cells (mSCARB2). We previously generated adapted strains (EV71:TLLm and EV71:TLLmv) that were shown to productively infect primate and rodent cell lines and whose genomes exhibited a multitude of non-synonymous mutations compared with the EV71:BS parental virus. In this study, we aimed to identify mutations that are necessary for productive infection of murine cells by EV71:BS. Using reverse genetics and site-directed mutagenesis, we constructed EV71 infectious clones with specific mutations that generated amino acid substitutions in the capsid VP1 and VP2 proteins. We subsequently assessed the infection induced by clone-derived viruses (CDVs) in mouse embryonic fibroblast NIH/3T3 and murine neuroblastoma Neuro-2a cell lines. We found that the CDV:BS-VP1(K98E,E145A,L169F) with three substitutions in the VP1 protein-K98E, E145A and L169F-productively infected both mouse cell lines for at least three passages of the virus in murine cells. Moreover, the virus gained the ability to utilize the mSCARB2 protein to infect murine cell lines. These results demonstrate that the three VP1 residues cooperate to effectively interact with the mSCARB2 protein on murine cells and permit the virus to infect murine cells. Gain-of-function studies similar to the present work provide valuable insight into the mutational trajectory required for EV71 to infect new host cells previously non-susceptible to infection.

  19. Comparative characteristics of three human embryonic stem cell lines.

    PubMed

    Lee, Jung Bok; Kim, Jin Mee; Kim, Sung Jong; Park, Jong Hyuk; Hong, Seok Ho; Roh, Sung Il; Kim, Moon Kyoo; Yoon, Hyun Soo

    2005-02-28

    Human embryonic stem (hES) cells have unique features including unlimited growth capacity, expression of specific markers, normal karyotypes and an ability to differentiate. Many investigators have tried to use hES cells for cell-based therapy, but there is little information about the properties of available hES cell lines. We compared the characteristics of three hES cell lines. The expression of SSEA-1, -3, -4, and APase, was examined by immunocytochemistry, and Oct-4 expression was analyzed by RT-PCR. Differentiation of the hES cells in vitro and in vivo led to the formation of embryoid bodies (EBs) or teratomas. We examined the expression of tissue-specific markers in the differentiated cells by semiquantitative RT-PCR, and the ability of each hES cell line to proliferate was measured by flow cytometry of DNA content and ELISA. The three hES cell lines were similar in morphology, marker expression, and teratoma formation. However there were significant differences (P < 0.05) between the differentiated cells formed by the different cell lines in levels of expression of tissue-specific markers such as renin, kallikrein, Glut-2, beta- and delta-globin, albumin, and alpha1-antitrypsin (alpha1-AT). The hES cell lines also differed in proliferative activity. Our observations should be useful in basic and clinical hES cell research.

  20. Identification of cell lines permissive for human coronavirus NL63.

    PubMed

    Schildgen, Oliver; Jebbink, Maarten F; de Vries, Michel; Pyrc, Krzysztov; Dijkman, Ronald; Simon, Arne; Müller, Andreas; Kupfer, Bernd; van der Hoek, Lia

    2006-12-01

    Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin-Darby-Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.

  1. Autoimmune damage to spermatogenesis in rodents immunized with mouse F9 embryonic carcinoma cells.

    PubMed Central

    Vojtiskova, M; Pokorna, Z; Draber, P

    1983-01-01

    Significant inhibition of spermatogenesis and appearance of antibodies against spermatogenic cells identified by cytotoxicity and immunofluorescence reactions were observed in mice of inbred strains 129/Sv and BALB/c and in albino guinea pigs after syngeneic, allogeneic, and xenogeneic immunization with mouse F9 embryonic carcinoma cells and Freund's complete adjuvant. A similar syngeneic immunization with PYS-2 cells was ineffective. Appropriate absorption experiments confirmed the similarity between the antigens of F9 and spermatogenic cells and the absence of such a similarity with antigens of PYS-2 cells. These results support the hypothesis that the oncofetal F9 antigens represent spermatogenic differentiation antigens and thus play an essential role in spermatogenic cell differentiation. PMID:6340100

  2. Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis

    PubMed Central

    Nichols, Nicole L.; Gowing, Genevieve; Satriotomo, Irawan; Nashold, Lisa J.; Dale, Erica A.; Suzuki, Masatoshi; Avalos, Pablo; Mulcrone, Patrick L.; McHugh, Jacalyn

    2013-01-01

    Rationale: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1G93A rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. Objectives: To preserve or restore phrenic nerve activity in SOD1G93A rats at disease end stage. Methods: SOD1G93A rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. Measurements and Main Results: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. Conclusions: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS. PMID:23220913

  3. Synergist Ablation as a Rodent Model to Study Satellite Cell Dynamics in Adult Skeletal Muscle.

    PubMed

    Kirby, Tyler J; McCarthy, John J; Peterson, Charlotte A; Fry, Christopher S

    2016-01-01

    In adult skeletal muscles, satellite cells are the primary myogenic stem cells involved in myogenesis. Normally, they remain in a quiescent state until activated by a stimulus, after which they proliferate, differentiate, and fuse into an existing myofiber or form a de novo myofiber. To study satellite cell dynamics in adult murine models, most studies utilize regeneration models in which the muscle is severely damaged and requires the participation from satellite cells in order to repair. Here, we describe a model to study satellite cell behavior in muscle hypertrophy that is independent of muscle regeneration.Synergist ablation surgery involves the surgical removal of the gastrocnemius and soleus muscles resulting in functional overload of the remaining plantaris muscle. This functional overload results in myofiber hypertrophy, as well as the activation, proliferation, and fusion of satellite cells into the myofibers. Within 2 weeks of functional overload, satellite cell content increases approximately 275 %, an increase that is accompanied with a ~60 % increase in the number of myonuclei. Therefore, this can be used as an alternative model to study satellite cell behavior in adulthood that is different from regeneration, and capable of revealing new satellite cell functions in regulating muscle adaptation.

  4. Espin cytoskeletal proteins in the sensory cells of rodent taste buds.

    PubMed

    Sekerková, Gabriella; Freeman, David; Mugnaini, Enrico; Bartles, James R

    2005-09-01

    Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 +/- 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 +/- 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP(3)R(3))(,) alpha-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP(3)R(3), PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP(3)R(3), alpha-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a

  5. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    Thevenin, E.; McMillian, J.

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  6. 1-Fluoro-2,4-dinitrobenzene and its derivatives act as secretagogues on rodent mast cells.

    PubMed

    Manabe, Yohei; Yoshimura, Marie; Sakamaki, Kazuma; Inoue, Asuka; Kakinoki, Aya; Hokari, Satoshi; Sakanaka, Mariko; Aoki, Junken; Miyachi, Hiroyuki; Furuta, Kazuyuki; Tanaka, Satoshi

    2017-01-01

    Accumulating evidence suggests that activated mast cells are involved in contact hypersensitivity, although the precise mechanisms of their activation are still not completely understood. We investigated the potential of common experimental allergens to induce mast cell activation using murine bone marrow-derived cultured mast cells and rat peritoneal mast cells. Among these allergens, 1-chloro-2,4-dinitrobenzene and 1-fluoro-2,4-dinirobenzene (DNFB) were found to induce degranulation of rat peritoneal mast cells. DNFB-induced degranulation is accompanied by cytosolic Ca(2+) mobilization and is significantly inhibited by pertussis toxin, U73122 (a phospholipase C inhibitor), and BAPTA (a Ca(2+) chelator), raising the possibility that DNFB acts on the G protein-coupled receptors and activates Gi , which induces activation of phospholipase C, as well as known mast cell secretagogues, such as compound 48/80. DNFB could induce mast cell degranulation in the absence of serum proteins and IgE. Structure-activity relationship analyses revealed an inverse correlation between the degree of degranulation and the electron density of the C1 carbon of the DNFB derivatives. These findings raise a possibility that DNFB functions as a potent contact allergen through induction of cutaneous mast cell degranulation.

  7. Apoptosis and proliferation of oligodendrocyte progenitor cells in the irradiated rodent spinal cord

    SciTech Connect

    Atkinson, Shelley L.; Li Yuqing; Wong, C. Shun . E-mail: shun.wong@sw.ca

    2005-06-01

    Purpose: Oligodendrocytes undergo early apoptosis after irradiation. The aim of this study was to determine the relationship between oligodendroglial apoptosis and proliferation of oligodendrocyte progenitor cells (OPC) in the irradiated central nervous system. Methods and Materials: Adult rats and p53 transgenic mice were given single doses of 2 Gy, 8 Gy, or 22 Gy to the cervical spinal cord. Apoptosis was assessed using TUNEL (Tdt-mediated dUTP terminal nick-end labeling) staining or by examining nuclear morphology. Oligodendrocyte progenitor cells were identified with an NG2 antibody or by in situ hybridization for platelet-derived growth factor receptor {alpha}. Proliferation of OPC was assessed by in vivo bromodeoxyuridine (BrdU) labeling and subsequent immunohistochemistry. Because radiation-induced apoptosis of oligodendroglial cells is p53 dependent, p53 transgenic mice were used to study the relationship between apoptosis and cell proliferation. Results: Oligodendrocyte progenitor cells underwent apoptosis within 24 h of irradiation in the rat. That did not result in a change in OPC density at 24 h. Oligodendrocyte progenitor cell density was significantly reduced by 2-4 weeks, but showed recovery by 6 weeks after irradiation. An increase in BrdU-labeled cells was observed at 2 weeks after 8 Gy or 22 Gy, and proliferating cells in the rat spinal cord were immunoreactive for NG2. The mouse spinal cord showed a similar early cell proliferation after irradiation. No difference was observed in the proliferation response in the spinal cord of p53 -/- mice compared with wild type animals. Conclusions: Oligodendroglial cells undergo early apoptosis and OPC undergo early proliferation after ionizing radiation. However, apoptosis is not likely to be the trigger for early proliferation of OPC in the irradiated central nervous system.

  8. Phenotype and Genotype of Pancreatic Cancer Cell Lines

    PubMed Central

    Deer, Emily L.; Gonzalez-Hernandez, Jessica; Coursen, Jill D.; Shea, Jill E.; Ngatia, Josephat; Scaife, Courtney L.; Firpo, Matthew A.; Mulvihill, Sean J.

    2009-01-01

    The dismal prognosis of pancreatic adenocarcinoma (PA) is due in part due to a lack of molecular information regarding disease development. Established cell lines remain a useful tool for investigating these molecular events. Here we present a review of available information on commonly used PA cell lines as a resource to help investigators select the cell lines most appropriate for their particular research needs. Information on clinical history, in vitro and in vivo growth characteristics, phenotypic characteristics, such as adhesion, invasion, migration and tumorigenesis, and genotypic status of commonly altered genes (KRAS, p53, p16, and SMAD4) was evaluated. Identification of both consensus and discrepant information in the literature suggests careful evaluation before selection of cell lines and attention be given to cell line authentication. PMID:20418756

  9. Motoneuron differentiation of immortalized human spinal cord cell lines.

    PubMed

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  10. GREG cells, a dysferlin-deficient myogenic mouse cell line

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; Morree, Antoine de; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  11. Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells

    PubMed Central

    Mazzara, Pietro Giuseppe; Massimino, Luca; Pellegatta, Marta; Ronchi, Giulia; Ricca, Alessandra; Iannielli, Angelo; Giannelli, Serena Gea; Cursi, Marco; Cancellieri, Cinzia; Sessa, Alessandro; Del Carro, Ubaldo; Quattrini, Angelo; Geuna, Stefano; Gritti, Angela; Taveggia, Carla; Broccoli, Vania

    2017-01-01

    Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies. PMID:28169300

  12. Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain.

    PubMed

    Hallmann, Anna-Lena; Araúzo-Bravo, Marcos J; Zerfass, Christina; Senner, Volker; Ehrlich, Marc; Psathaki, Olympia E; Han, Dong Wook; Tapia, Natalia; Zaehres, Holm; Schöler, Hans R; Kuhlmann, Tanja; Hargus, Gunnar

    2016-05-01

    Reprogramming technology enables the production of neural progenitor cells (NPCs) from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs) differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs) and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs.

  13. Pharmacologic Stem Cell Based Intervention as a New Approach to Osteoporosis Treatment in Rodents

    PubMed Central

    Bi, Yanming; Liu, Yongzhong; Akiyama, Kentaro; Sonoyama, Wataru; Patel, Voymesh; Gutkind, Silvio; Young, Marian; Gronthos, Stan; Le, Anh; Wang, Cun-Yu; Chen, WanJun; Shi, Songtao

    2008-01-01

    Background Osteoporosis is the most prevalent skeletal disorder, characterized by a low bone mineral density (BMD) and bone structural deterioration, leading to bone fragility fractures. Accelerated bone resorption by osteoclasts has been established as a principal mechanism in osteoporosis. However, recent experimental evidences suggest that inappropriate apoptosis of osteoblasts/osteocytes accounts for, at least in part, the imbalance in bone remodeling as occurs in osteoporosis. The aim of this study is to examine whether aspirin, which has been reported as an effective drug improving bone mineral density in human epidemiology studies, regulates the balance between bone resorption and bone formation at stem cell levels. Methods and Findings We found that T cell-mediated bone marrow mesenchymal stem cell (BMMSC) impairment plays a crucial role in ovariectomized-induced osteoporosis. Ex vivo mechanistic studies revealed that T cell-mediated BMMSC impairment was mainly attributed to the apoptosis of BMMSCs via the Fas/Fas ligand pathway. To explore potential of using pharmacologic stem cell based intervention as an approach for osteoporosis treatment, we selected ovariectomy (OVX)-induced ostoeporosis mouse model to examine feasibility and mechanism of aspirin-mediated therapy for osteoporosis. We found that aspirin can inhibit T cell activation and Fas ligand induced BMMSC apoptosis in vitro. Further, we revealed that aspirin increases osteogenesis of BMMSCs by aiming at telomerase activity and inhibits osteoclast activity in OVX mice, leading to ameliorating bone density. Conclusion Our findings have revealed a novel osteoporosis mechanism in which activated T cells induce BMMSC apoptosis via Fas/Fas ligand pathway and suggested that pharmacologic stem cell based intervention by aspirin may be a new alternative in osteoporosis treatment including activated osteoblasts and inhibited osteoclasts. PMID:18612428

  14. Proteasome Dysfunction Mediates High Glucose-Induced Apoptosis in Rodent Beta Cells and Human Islets

    PubMed Central

    Broca, Christophe; Varin, Elodie; Armanet, Mathieu; Tourrel-Cuzin, Cécile; Bosco, Domenico; Dalle, Stéphane; Wojtusciszyn, Anne

    2014-01-01

    The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic beta cells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E beta cells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3-dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized beta cells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes. PMID:24642635

  15. Germ line, stem cells, and epigenetic reprogramming.

    PubMed

    Surani, M A; Durcova-Hills, G; Hajkova, P; Hayashi, K; Tee, W W

    2008-01-01

    The germ cell lineage has the unique attribute of generating the totipotent state. Development of blastocysts from the totipotent zygote results in the establishment of pluripotent primitive ectoderm cells in the inner cell mass of blastocysts, which subsequently develop into epiblast cells in postimplantation embryos. The germ cell lineage in mice originates from these pluripotent epiblast cells of postimplantation embryos in response to specific signals. Pluripotent stem cells and unipotent germ cells share some fundamental properties despite significant phenotypic differences between them. Additionally, early primordial germ cells can be induced to undergo dedifferentiation into pluripotent embryonic germ cells. Investigations on the relationship between germ cells and pluripotent stem cells may further elucidate the nature of the pluripotent state. Furthermore, comprehensive epigenetic reprogramming of the genome in early germ cells, including extensive erasure of epigenetic modifications, is a critical step toward establishment of totipotency. The mechanisms involved may be relevant for gaining insight into events that lead to reprogramming of somatic cells into pluripotent stem cells.

  16. Ectopic Purkinje cells in the cerebellar white matter of normal adult rodents: a Golgi study.

    PubMed

    Lafarga, M; Berciano, M T; Blanco, M

    1986-01-01

    In Golgi/Río-Hortega preparations of rat and rabbit cerebellar vermis we have occasionally found isolated ectopic Purkinje cells in the white matter. They were located beneath the bases of the folia and their dendritic branches extended within the confines of the white matter without penetrating into the overlying cortical layers. The general morphology of these ectopic cells was variable, particularly in the extension and shape of the dendritic trees, but all of them exhibited a lower density of dendritic branches than normal Purkinje cells. The less-developed ectopic neurons had multipolar dendritic trees with nonplanar branches irregularly studded with spines. The well-developed ones displayed a more extensive arborization of their processes and they usually preserved some morphological features of normal cortical Purkinje cells: distal dendritic branches studded with numerous spines, a pear-shaped soma, clearly defined morphological polarity and a tendency to display planar arrangement of the dendritic arbors. In semithin sections these neurons also showed cytological features of normal Purkinje cells, such as the Nissl substance forming a nuclear cap oriented toward the dendritic pole. We suggest that the abnormal location of the neurons results from a disorder of Purkinje cell migration which occurs naturally during the prenatal development of the cerebellum. The possible morphogenetic mechanisms involved in the migration and differentiation of these ectopic neurons are also discussed.

  17. West Nile virus infection does not induce PKR activation in rodent cells.

    PubMed

    Elbahesh, H; Scherbik, S V; Brinton, M A

    2011-12-05

    dsRNA-activated protein kinase (PKR) is activated by viral dsRNAs and phosphorylates eIF2a reducing translation of host and viral mRNA. Although infection with a chimeric West Nile virus (WNV) efficiently induced PKR and eIF2a phosphorylation, infections with natural lineage 1 or 2 strains did not. Investigation of the mechanism of suppression showed that among the cellular PKR inhibitor proteins tested, only Nck, known to interact with inactive PKR, colocalized and co-immunoprecipitated with PKR in WNV-infected cells and PKR phosphorylation did not increase in infected Nck1,2-/- cells. Several WNV stem-loop RNAs efficiently activated PKR in vitro but not in infected cells. WNV infection did not interfere with intracellular PKR activation by poly(I:C) and similar virus yields were produced by control and PKR-/- cells. The results indicate that PKR phosphorylation is not actively suppressed in WNV-infected cells but that PKR is not activated by the viral dsRNA in infected cells.

  18. West Nile virus infection does not induce PKR activation in rodent cells

    PubMed Central

    Elbahesh, H.; Scherbik, S. V.; Brinton, M. A.

    2011-01-01

    dsRNA-activated protein kinase (PKR) is activated by viral dsRNAs and phosphorylates eIF2a reducing translation of host and viral mRNA. Although infection with a chimeric West Nile virus (WNV) efficiently induced PKR and eIF2a phosphorylation, infections with natural lineage 1 or 2 strains did not. Investigation of the mechanism of suppression showed that among the cellular PKR inhibitor proteins tested, only Nck, known to interact with inactive PKR, colocalized and co-immunoprecipitated with PKR in WNV-infected cells and PKR phosphorylation did not increase in infected Nck1,2−/− cells. Several WNV stem-loop RNAs efficiently activated PKR in vitro but not in infected cells. WNV infection did not interfere with intracellular PKR activation by poly(I:C) and similar virus yields were produced by control and PKR−/− cells. The results indicate that PKR phosphorylation is not actively suppressed in WNV-infected cells but that PKR is not activated by the viral dsRNA in infected cells. PMID:21982595

  19. Establishment of a Human Thymic Myoid Cell Line

    PubMed Central

    Wakkach, Abdel; Poea, Sandrine; Chastre, Eric; Gespach, Christian; Lecerf, Florence; De la Porte, Sabine; Tzartos, Socrates; Coulombe, Alain; Berrih-Aknin, Sonia

    1999-01-01

    The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. α-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells. PMID:10514405

  20. Deriving cell lines from zebrafish embryos and tumors.

    PubMed

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

    2013-09-01

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

  1. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Functional calcium imaging in zebrafish lateral-line hair cells.

    PubMed

    Zhang, Q X; He, X J; Wong, H C; Kindt, K S

    2016-01-01

    Sensory hair-cell development, function, and regeneration are fundamental processes that are challenging to study in mammalian systems. Zebrafish are an excellent alternative model to study hair cells because they have an external auxiliary organ called the lateral line. The hair cells of the lateral line are easily accessible, which makes them suitable for live, function-based fluorescence imaging. In this chapter, we describe methods to perform functional calcium imaging in zebrafish lateral-line hair cells. We compare genetically encoded calcium indicators that have been used previously to measure calcium in lateral-line hair cells. We also outline equipment required for calcium imaging and compare different imaging systems. Lastly, we discuss how to set up optimal imaging parameters and how to process and visualize calcium signals. Overall, using these methods, in vivo calcium imaging is a powerful tool to examine sensory hair-cell function in an intact organism. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors

    PubMed Central

    Hu, Peirong; Li, Yedda; Sands, Mark S; McCown, Thomas; Kafri, Tal

    2015-01-01

    The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~107 infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 108 IU/mL, which upon concentration increased to 1010 IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications. PMID:26229972

  4. Generation of a stable packaging cell line producing high-titer PPT-deleted integration-deficient lentiviral vectors.

    PubMed

    Hu, Peirong; Li, Yedda; Sands, Mark S; McCown, Thomas; Kafri, Tal

    2015-01-01

    The risk of insertional mutagenesis inherent to all integrating exogenous expression cassettes was the impetus for the development of various integration-defective lentiviral vector (IDLV) systems. These systems were successfully employed in a plethora of preclinical applications, underscoring their clinical potential. However, current production of IDLVs by transient plasmid transfection is not optimal for large-scale production of clinical grade vectors. Here, we describe the development of the first tetracycline-inducible stable IDLV packaging cell line comprising the D64E integrase mutant and the VSV-G envelope protein. A conditional self-inactivating (cSIN) vector and a novel polypurine tract (PPT)-deleted vector were incorporated into the newly developed stable packaging cell line by transduction and stable transfection, respectively. High-titer (~10(7) infectious units (IU)/ml) cSIN vectors were routinely generated. Furthermore, screening of single-cell clones stably transfected with PPT-deleted vector DNA resulted in the identification of highly efficient producer cell lines generating IDLV titers higher than 10(8) IU/mL, which upon concentration increased to 10(10) IU/ml. IDLVs generated by stable producer lines efficiently transduce CNS tissues of rodents. Overall, the availability of high-titer IDLV lentivirus packaging cell line described here will significantly facilitate IDLV-based basic science research, as well as preclinical and clinical applications.

  5. High Density Individually Addressable Nanowire Arrays Record Intracellular Activity from Primary Rodent and Human Stem Cell Derived Neurons.

    PubMed

    Liu, Ren; Chen, Renjie; Elthakeb, Ahmed T; Lee, Sang Heon; Hinckley, Sandy; Khraiche, Massoud L; Scott, John; Pre, Deborah; Hwang, Yoontae; Tanaka, Atsunori; Ro, Yun Goo; Matsushita, Albert K; Dai, Xing; Soci, Cesare; Biesmans, Steven; James, Anthony; Nogan, John; Jungjohann, Katherine L; Pete, Douglas V; Webb, Denise B; Zou, Yimin; Bang, Anne G; Dayeh, Shadi A

    2017-04-10

    We report a new hybrid integration scheme that offers for the first time a nanowire-on-lead approach, which enables independent electrical addressability, is scalable, and has superior spatial resolution in vertical nanowire arrays. The fabrication of these nanowire arrays is demonstrated to be scalable down to submicrometer site-to-site spacing and can be combined with standard integrated circuit fabrication technologies. We utilize these arrays to perform electrophysiological recordings from mouse and rat primary neurons and human induced pluripotent stem cell (hiPSC)-derived neurons, which revealed high signal-to-noise ratios and sensitivity to subthreshold postsynaptic potentials (PSPs). We measured electrical activity from rodent neurons from 8 days in vitro (DIV) to 14 DIV and from hiPSC-derived neurons at 6 weeks in vitro post culture with signal amplitudes up to 99 mV. Overall, our platform paves the way for longitudinal electrophysiological experiments on synaptic activity in human iPSC based disease models of neuronal networks, critical for understanding the mechanisms of neurological diseases and for developing drugs to treat them.

  6. Generation of mesenchymal stem cell lines from murine bone marrow.

    PubMed

    Sreejit, P; Dilip, K B; Verma, R S

    2012-10-01

    Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.

  7. Gastrin gene expression and regulation in rat islet cell lines.

    PubMed

    Brand, S J; Wang, T C

    1988-11-15

    Gastrin gene expression was observed in two permanent rat insulinoma (RIN) cell lines derived from a rat insulinoma. Gastrin expression was selective; highest expression was seen in a cell line which did not express other islet cell hormones. Gastrin mRNA transcription initiated from the same promoter as antral gastrin mRNA. DNA transfection studies with a gastrin chloramphenicol acetyltransferase chimeric gene showed higher expression in gastrin-expressing RIN cells than non-gastrin-expressing islet cells. This implies that gastrin-expressing RIN cells selectively express a trans-acting transcriptional activator which binds to cis-acting regulatory sequences within the 5'-flanking DNA sequence and first exon of the gastrin gene. The gastrin peptide precursor synthesized in these RIN cell lines is subject to the same repertoire of posttranslational modifications within the cell's secretory apparatus (endoproteolytic cleavage, tyrosine sulfation, and C-terminal amidation) as seen in antral G cells. Gastrin mRNA levels in these RIN cells were selectively increased by increasing the extracellular calcium concentration. Membrane depolarization also stimulated gastrin mRNA levels, probably through activation of voltage-sensitive calcium channels. Thus, these gastrin-expressing RIN cell lines provide permanent cell lines useful in analyzing the cellular regulation of gastrin gene expression.

  8. Quantitative methods to characterize morphological properties of cell lines.

    PubMed

    Mancia, Annalaura; Elliott, John T; Halter, Michael; Bhadriraju, Kiran; Tona, Alessandro; Spurlin, Tighe A; Middlebrooks, Bobby L; Baatz, John E; Warr, Gregory W; Plant, Anne L

    2012-07-01

    Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages.

  9. Anthocyanins do not influence long-chain n-3 fatty acid status: studies in cells, rodents and humans☆

    PubMed Central

    Vauzour, David; Tejera, Noemi; O'Neill, Colette; Booz, Valeria; Jude, Baptiste; Wolf, Insa M.A.; Rigby, Neil; Silvan, Jose Manuel; Curtis, Peter J.; Cassidy, Aedin; de Pascual-Teresa, Sonia; Rimbach, Gerald; Minihane, Anne Marie

    2015-01-01

    Increased tissue status of the long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) is associated with cardiovascular and cognitive benefits. Limited epidemiological and animal data suggest that flavonoids, and specifically anthocyanins, may increase EPA and DHA levels, potentially by increasing their synthesis from the shorter-chain n-3 PUFA, α-linolenic acid. Using complimentary cell, rodent and human studies we investigated the impact of anthocyanins and anthocyanin-rich foods/extracts on plasma and tissue EPA and DHA levels and on the expression of fatty acid desaturase 2 (FADS2), which represents the rate limiting enzymes in EPA and DHA synthesis. In experiment 1, rats were fed a standard diet containing either palm oil or rapeseed oil supplemented with pure anthocyanins for 8 weeks. Retrospective fatty acid analysis was conducted on plasma samples collected from a human randomized controlled trial where participants consumed an elderberry extract for 12 weeks (experiment 2). HepG2 cells were cultured with α-linolenic acid with or without select anthocyanins and their in vivo metabolites for 24 h and 48 h (experiment 3). The fatty acid composition of the cell membranes, plasma and liver tissues were analyzed by gas chromatography. Anthocyanins and anthocyanin-rich food intake had no significant impact on EPA or DHA status or FADS2 gene expression in any model system. These data indicate little impact of dietary anthocyanins on n-3 PUFA distribution and suggest that the increasingly recognized benefits of anthocyanins are unlikely to be the result of a beneficial impact on tissue fatty acid status. PMID:25573539

  10. Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells

    PubMed Central

    Lecht, Shimon; Stabler, Collin T.; Rylander, Alexis L.; Chiaverelli, Rachel; Schulman, Edward S.; Marcinkiewicz, Cezary; Lelkes, Peter I.

    2016-01-01

    Repopulation of decellularized lung scaffolds (DLS) is limited due to alterations in the repertoire and ratios of the residual extracellular matrix (ECM) proteins, characterized by e.g., the retention of type I collagen and loss of glycoproteins. We hypothesized that pre-treatment of decellularized matrices with defined ECM proteins, which match the repertoire of integrin receptors expressed by the cells to be seeded (e.g., embryonic stem cells) can increase the efficacy of the reseeding process. To test this hypothesis, we first determined the integrin receptors profile of mouse embryonic stem cells (mESCs). Mouse ESCs express α3, α5, α6, α9 and β1, but not α1, α2 and α4 integrin subunits, as established by Western blotting and adhesion to laminin and fibronectin, but not to collagens type I and IV. Reseeding of DLS with mESCs was inefficient (6.9 ± 0.5%), but was significantly enhanced (2.3 ± 0.1 fold) by pre-treating the scaffolds with media conditioned by A549 human lung adenocarcinoma cells, which we found to contain ~5 μg/ml laminin. Furthermore, pre-treatment with A549-conditioned media resulted in a significantly more uniform distribution of the seeded mESCs throughout the engineered organ as compared to untreated DLS. Our study may advance whole lung engineering by stressing the importance of matching the integrin receptor repertoire of the seeded cells and the cell binding motifs of DLS. PMID:24439414

  11. Cell line models for differentiation: preadipocytes and adipocytes.

    PubMed

    Poulos, Sylvia P; Dodson, Michael V; Hausman, Gary J

    2010-10-01

    In vitro models have been invaluable in determining the mechanisms involved in adipocyte proliferation, differentiation, adipokine secretion and gene/protein expression. The cells presently available for research purposes all have unique advantages and disadvantages that one should be aware of when selecting cells. Established cell lines, such as 3T3-L1 cells, are easier and less costly to use than freshly isolated cells, even though freshly isolated cells allow for various comparisons such as the in vitro evaluation of different in vivo conditions that may not be possible using cell lines. Moreover, stem cells, transdifferentiated cells or dedifferentiated cells are relatively new cell models being evaluated for the study of adipocyte regulation and physiology. The focus of this brief review is to highlight similarities and differences in adipocyte models to aid in appropriate model selection and data interpretation for successful advancement of our understanding of adipocyte biology.

  12. Dietary Zinc Deficiency in Rodents: Effects on T-Cell Development, Maturation and Phenotypes

    PubMed Central

    Blewett, Heather J.; Taylor, Carla G.

    2012-01-01

    Zinc deficiency is one of the leading risk factors for developing disease and yet we do not have a clear understanding of the mechanisms behind the increased susceptibility to infection. This review will examine the interrelationships among the hypothalamus-pituitary-adrenal stress axis, p56lck, and T-cell maturation in both zinc deficiency and responses during zinc repletion. We will highlight differences between the adult mouse model (wasting malnutrition) and growing rat model (stunting malnutrition) of dietary zinc deficiency and discuss the use of various controls to separate out the effects of zinc deficiency from the associated malnutrition. Elevated serum corticosterone in both zinc deficient and pair-fed rats does not support the hypothesis that zinc deficiency per se leads to corticosterone-induced apoptosis and lymphopenia. In fact, the zinc deficient rat does not have lymphopenia. Thymocytes from zinc deficient mice and rats have elevated levels of p56lck, a signalling protein with a zinc clasp structure, but this does not appear to affect thymocyte maturation. However, post-thymic T-cell maturation appears to be altered based on the lower proportion of splenic late thymic emigrants in zinc deficient rats. Fewer new T-cells in the periphery could adversely affect the T-cell repertoire and contribute to immunodeficiency in zinc deficiency. PMID:22822446

  13. cDNA cloning and expression of HIP, a novel cell surface heparan sulfate/heparin-binding protein of human uterine epithelial cells and cell lines.

    PubMed

    Liu, S; Smith, S E; Julian, J; Rohde, L H; Karin, N J; Carson, D D

    1996-05-17

    Heparan sulfate proteoglycans and their corresponding binding sites have been suggested to play an important role during the initial attachment of murine blastocysts to uterine epithelium and human trophoblastic cell lines to uterine epithelial cell lines. Previous studies on RL95 cells, a human uterine epithelial cell line, had characterized a single class of cell surface heparin/heparan sulfate (HP/HS)-binding sites. Three major HP/HS-binding peptide fragments were isolated from cell surfaces by tryptic digestion, and partial amino-terminal amino acid sequence for each peptide fragment was obtained (Raboudi, N., Julian, J., Rohde, L. H., and Carson, D. D. (1992) J. Biol. Chem. 267, 11930-11939). In the current study, using approaches of reverse transcription-polymerase chain reaction and cDNA library screening, we have cloned and expressed a novel, cell surface HP/HS-binding protein, named HP/HS interacting protein (HIP), from RL95 cells. The full-length cDNA of HIP encodes a protein of 159 amino acids with a calculated molecular mass of 17,754 Da and pI of 11.75. Transfection of HIP full-length cDNA into NIH-3T3 cells demonstrated cell surface expression and a size similar to that of HIP expressed by human cells. Predicted amino acid sequence indicates that HIP lacks a membrane spanning region and has no consensus sites for glycosylation. Northern blot analysis detected a single transcript of 1.3 kilobases in both total RNA and poly(A+) RNA. Examination of human cell lines and normal tissues using both Northern blot and Western blot analyses revealed that HIP is expressed at different levels in a variety of human cell lines and normal tissues but absent in some cell lines and some cell types of normal tissues examined. HIP has relatively high homology (approximately 80% both at the levels of nucleotide and protein sequence) to a rodent ribosomal protein L29. Thus, members of the L29 family may be displayed on cell surfaces where they may participate in HP

  14. Antineoplastic activity of rinvanil and phenylacetylrinvanil in leukaemia cell lines

    PubMed Central

    LUVIANO, AXEL; AGUIÑIGA-SÁNCHEZ, ITZEN; DEMARE, PATRICIA; TIBURCIO, REYNALDO; LEDESMA-MARTÍNEZ, EDGAR; SANTIAGO-OSORIO, EDELMIRO; REGLA, IGNACIO

    2014-01-01

    In the search for novel chemotherapeutic agents for cancer treatment, capsaicin has been shown to inhibit proliferation and induce apoptosis in various types of cancer cell line, including leukaemia cell lines. The capsaicin analogues, rinvanil and phenylacetylrinvanil (PhAR), share a binding affinity for vanilloid receptors and may have biological activities similar to capsaicin; however, their anticancer potential has not yet been reported. This study analyses the antineoplastic activities of rinvanil and PhAR in leukaemia versus normal cells. P388, J774 and WEHI-3 leukaemia cell lines, as well as mouse bone marrow mononuclear cells, were cultured with varying concentrations of rinvanil and PhAR. Following this, proliferation and apoptosis were determined by the sulforhodamine B (SRB) assay and DNA ladder. Cultured leukaemia cell lines and mouse bone marrow mononuclear cells demonstrated a dose-dependent inhibition of proliferation, while non-diseased cells were less sensitive to the cytotoxic effect of capsaicin, rinvanil and PhAR. Rinvanil and PhAR also induced apoptosis in leukaemia cell lines but not in bone marrow. Given the lower IC50 values for apoptosis induction in leukaemia cells compared with that of normal cells, PhAR is a promising selective anticancer agent. PMID:24765194

  15. Generation and sequencing of pulmonary carcinoid tumor cell lines.

    PubMed

    Asiedu, Michael K; Thomas, Charles F; Tomaszek, Sandra C; Peikert, Tobias; Sanyal, Bharati; Sutor, Shari L; Aubry, Marie-Christine; Li, Peter; Wigle, Dennis A

    2014-12-01

    Pulmonary carcinoid tumors account for approximately 5% of all lung malignancies in adults, and comprise 30% of all carcinoid tumors. There are limited reagents available to study these rare tumors, and consequently no major advances have been made for patient treatment. We report the generation and characterization of human pulmonary carcinoid tumor cell lines to study underlying biology, and to provide models for testing novel chemotherapeutic agents. Tissue was harvested from three patients with primary pulmonary typical carcinoid tumors undergoing surgical resection. The tumor was dissociated and plated onto dishes in culture media. The established cell lines were characterized by immunohistochemistry, Western blotting, and cell proliferation assays. Tumorigenicity was confirmed by soft agar growth and the ability to form tumors in a mouse xenograft model. Exome and RNA sequencing of patient tumor samples and cell lines was performed using standard protocols. Three typical carcinoid tumor lines grew as adherent monolayers in vitro, expressed neuroendocrine markers consistent with the primary tumor, and formed colonies in soft agar. A single cell line produced lung tumors in nude mice after intravenous injection. Exome and RNA sequencing of this cell line showed lineage relationship with the primary tumor, and demonstrated mutations in a number of genes related to neuronal differentiation. Three human pulmonary typical carcinoid tumor cell lines have been generated and characterized as a tool for studying the biology and novel treatment approaches for these rare tumors.

  16. Growth of Murine Cytomegalovirus in Various Cell Lines

    PubMed Central

    Kim, Kwang Soo; Carp, Richard I.

    1971-01-01

    Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV. PMID:4327583

  17. Neural representation of spatial topology in the rodent hippocampus.

    PubMed

    Chen, Zhe; Gomperts, Stephen N; Yamamoto, Jun; Wilson, Matthew A

    2014-01-01

    Pyramidal cells in the rodent hippocampus often exhibit clear spatial tuning in navigation. Although it has been long suggested that pyramidal cell activity may underlie a topological code rather than a topographic code, it remains unclear whether an abstract spatial topology can be encoded in the ensemble spiking activity of hippocampal place cells. Using a statistical approach developed previously, we investigate this question and related issues in greater detail. We recorded ensembles of hippocampal neurons as rodents freely foraged in one- and two-dimensional spatial environments and used a "decode-to-uncover" strategy to examine the temporally structured patterns embedded in the ensemble spiking activity in the absence of observed spatial correlates during periods of rodent navigation or awake immobility. Specifically, the spatial environment was represented by a finite discrete state space. Trajectories across spatial locations ("states") were associated with consistent hippocampal ensemble spiking patterns, which were characterized by a state transition matrix. From this state transition matrix, we inferred a topology graph that defined the connectivity in the state space. In both one- and two-dimensional environments, the extracted behavior patterns from the rodent hippocampal population codes were compared against randomly shuffled spike data. In contrast to a topographic code, our results support the efficiency of topological coding in the presence of sparse sample size and fuzzy space mapping. This computational approach allows us to quantify the variability of ensemble spiking activity, examine hippocampal population codes during off-line states, and quantify the topological complexity of the environment.

  18. Metabolic Profiling of Dividing Cells in Live Rodent Brain by Proton Magnetic Resonance Spectroscopy (1HMRS) and LCModel Analysis

    PubMed Central

    Park, June-Hee; Lee, Hedok; Makaryus, Rany; Yu, Mei; Smith, S. David; Sayed, Kasim; Feng, Tian; Holland, Eric; Van der Linden, Annemie; Bolwig, Tom G.; Enikolopov, Grigori; Benveniste, Helene

    2014-01-01

    Rationale Dividing cells can be detected in the live brain by positron emission tomography or optical imaging. Here we apply proton magnetic resonance spectroscopy (1HMRS) and a widely used spectral fitting algorithm to characterize the effect of increased neurogenesis after electroconvulsive shock in the live rodent brain via spectral signatures representing mobile lipids resonating at ∼1.30 ppm. In addition, we also apply the same 1HMRS methodology to metabolically profile glioblastomas with actively dividing cells growing in RCAS-PDGF mice. Methods 1HMRS metabolic profiles were acquired on a 9.4T MRI instrument in combination with LCModel spectral analysis of: 1) rat brains before and after ECS or sham treatments and 2) RCAS-PDGF mice with glioblastomas and wild-type controls. Quantified 1HMRS data were compared to post-mortem histology. Results Dividing cells in the rat hippocampus increased ∼3-fold after ECS compared to sham treatment. Quantification of hippocampal metabolites revealed significant decreases in N-acetyl-aspartate but no evidence of an elevated signal at ∼1.3 ppm (Lip13a+Lip13b) in the ECS compared to the sham group. In RCAS-PDGF mice a high density (22%) of dividing cells characterized glioblastomas. Nile Red staining revealed a small fraction (3%) of dying cells with intracellular lipid droplets in the tumors of RCAS-PDGF mice. Concentrations of NAA were lower, whereas lactate and Lip13a+Lip13b were found to be significantly higher in glioblastomas of RCAS-PDGF mice, when compared to normal brain tissue in the control mice. Conclusions Metabolic profiling using 1HMRS in combination with LCModel analysis did not reveal correlation between Lip13a+Lip13b spectral signatures and an increase in neurogenesis in adult rat hippocampus after ECS. However, increases in Lip13a+Lip13b were evident in glioblastomas suggesting that a higher density of actively dividing cells and/or the presence of lipid droplets is necessary for LCModel to reveal

  19. Investigation of Radiosensitivity Gene Signatures in Cancer Cell Lines

    PubMed Central

    Hall, John S.; Iype, Rohan; Senra, Joana; Taylor, Janet; Armenoult, Lucile; Oguejiofor, Kenneth; Li, Yaoyong; Stratford, Ian; Stern, Peter L.; O’Connor, Mark J.; Miller, Crispin J.; West, Catharine M. L.

    2014-01-01

    Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins. PMID:24466029

  20. The insulin/Akt pathway controls a specific cell division program that leads to generation of binucleated tetraploid liver cells in rodents

    PubMed Central

    Celton-Morizur, Séverine; Merlen, Grégory; Couton, Dominique; Margall-Ducos, Germain; Desdouets, Chantal

    2009-01-01

    The formation of polyploid cells is part of the developmental program of several tissues. During postnatal development, binucleated tetraploid cells arise in the liver, caused by failure in cytokinesis. In this report, we have shown that the initiation of cytokinesis failure events and the subsequent appearance of binucleated tetraploid cells are strictly controlled by the suckling-to-weaning transition in rodents. We found that daily light/dark rhythms and carbohydrate intake did not affect liver tetraploidy. In contrast, impairment of insulin signaling drastically reduced the formation of binucleated tetraploid cells, whereas repeated insulin injections promoted the generation of these liver cells. Furthermore, inhibition of Akt activity decreased the number of cytokinesis failure events, possibly through the mammalian target of rapamycin signaling complex 2 (mTORC2), which indicates that the PI3K/Akt pathway lies downstream of the insulin signal to regulate the tetraploidization process. To our knowledge, these results are the first demonstration in a physiological context that insulin signaling through Akt controls a specific cell division program and leads to the physiologic generation of binucleated tetraploid liver cells. PMID:19603546

  1. Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells

    PubMed Central

    Kovach, AnneMarie Kay; Gambino, Jen M.; Nguyen, Vina; Nelson, Zach; Szasz, Taylor; Liao, Jun; Williams, Lakiesha; Bulla, Sandra; Prabhu, Raj

    2016-01-01

    Abstract Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF) antibody and ligand cluster of differentiation 80 (CD80) for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA). The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTMNPO CRL-2836) at four different concentrations (0.1, 1.0, 10.0, and 100.0 μg/mL) of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF). Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 μg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 μg/mL of CD80+VEGF and 0.1 μg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 μg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 μg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test) induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum species

  2. Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells.

    PubMed

    Kovach, AnneMarie Kay; Gambino, Jen M; Nguyen, Vina; Nelson, Zach; Szasz, Taylor; Liao, Jun; Williams, Lakiesha; Bulla, Sandra; Prabhu, Raj

    2016-01-01

    Target drug deliveries using nanotechnology are a novel consideration in the treatment of cancer. We present herein an in vitro mouse model for the preliminary investigation of the efficacy of an iron oxide nanoparticle complex conjugated to vascular endothelial growth factor (VEGF) antibody and ligand cluster of differentiation 80 (CD80) for the purpose of eventual translational applications in the treatment of human osteosarcoma (OSA). The 35 nm diameter iron oxide magnetic nanoparticles are functionalized with an n-hydroxysuccinimide biocompatible coating and are conjugated on the surface to proteins VEGF antibody and ligand CD80. Combined, these proteins have the ability to target OSA cells and induce apoptosis. The proposed system was tested on a cancerous rodent osteoblast cell line (ATCCTM(NPO) CRL-2836) at four different concentrations (0.1, 1.0, 10.0, and 100.0 μg/mL) of ligand CD80 alone, VEGF antibody alone, and a combination thereof (CD80+VEGF). Systems were implemented every 24 h over different sequential treatment timelines: 24, 48, and 72 h, to find the optimal protein concentration required for a reduction in cell proliferation. Results demonstrated that a combination of ligand CD80 and VEGF antibody was consistently most effective at reducing aberrant osteoblastic proliferation for both the 24- and 72-h timelines. At 48 h, however, an increase in cell proliferation was documented for the 0.1 and 1 μg/mL groups. For the 24- and 72-h tests, concentrations of 1.0 μg/mL of CD80+VEGF and 0.1 μg/mL of VEGF antibody were most effective. Concentrations of 10.0 and 100.0 μg/mL of CD80+VEGF reduced cell proliferation, but not as remarkably as the 1.0 μg/mL concentration. In addition, cell proliferation data showed that multiple treatments (72-h test) induced cell death in the osteoblasts better than a single treatment. Future targeted drug delivery system research includes trials in OSA cell lines from greater phylum species having

  3. Genotoxic Changes to Rodent Cells Exposed in Vitro to Tungsten, Nickel, Cobalt and Iron

    DTIC Science & Technology

    2014-03-10

    available gene chips. The results obtained were compared to untreated controls and tantalum -treated cells. Tantalum has been shown to be an inert non...serum mutagenicity studies with rats implanted with depleted uranium or tantalum pellets. Mutagenesis 1998, 13, 643–648. 6. McClain, D.E.; Benson, K.A...bone and tantalum , niobium or commercially pure titanium. Biomaterials 1990, 11, 277–280. 32. Fan, J.B.; Gunderson, K.L.; Bibikova, M.; Yeakley, J.M

  4. Regulation of germ line stem cell homeostasis

    PubMed Central

    Garcia, T.X.; Hofmann, M.C.

    2015-01-01

    Mammalian spermatogenesis is a complex process in which spermatogonial stem cells of the testis (SSCs) develop to ultimately form spermatozoa. In the seminiferous epithelium, SSCs self-renew to maintain the pool of stem cells throughout life, or they differentiate to generate a large number of germ cells. A balance between SSC self-renewal and differentiation is therefore essential to maintain normal spermatogenesis and fertility. Stem cell homeostasis is tightly regulated by signals from the surrounding microenvironment, or SSC niche. By physically supporting the SSCs and providing them with these extrinsic molecules, the Sertoli cell is the main component of the niche. Earlier studies have demonstrated that GDNF and CYP26B1, produced by Sertoli cells, are crucial for self-renewal of the SSC pool and maintenance of the undifferentiated state. Down-regulating the production of these molecules is therefore equally important to allow germ cell differentiation. We propose that NOTCH signaling in Sertoli cells is a crucial regulator of germ cell fate by counteracting these stimulatory factors to maintain stem cell homeostasis. Dysregulation of this essential niche component can lead by itself to sterility or facilitate testicular cancer development.

  5. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions

    NASA Astrophysics Data System (ADS)

    Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

  6. Karyotype alteration generates the neoplastic phenotypes of SV40-infected human and rodent cells.

    PubMed

    Bloomfield, Mathew; Duesberg, Peter

    2015-01-01

    Despite over 50 years of research, it remains unclear how the DNA tumor viruses SV40 and Polyoma cause cancers. Prevailing theories hold that virus-coded Tumor (T)-antigens cause cancer by inactivating cellular tumor suppressor genes. But these theories don't explain four characteristics of viral carcinogenesis: (1) less than one in 10,000 infected cells become cancer cells, (2) cancers have complex individual phenotypes and transcriptomes, (3) recurrent tumors without viral DNA and proteins, (4) preneoplastic aneuploidies and immortal neoplastic clones with individual karyotypes. As an alternative theory we propose that viral carcinogenesis is a form of speciation, initiated by virus-induced aneuploidy. Since aneuploidy destabilizes the karyotype by unbalancing thousands of genes it catalyzes chain reactions of karyotypic and transcriptomic evolutions. Eventually rare karyotypes evolve that encode cancer-specific autonomy of growth. The low probability of forming new autonomous cancer-species by random karyotypic and transcriptomic variations predicts individual and clonal cancers. Although cancer karyotypes are congenitally aneuploid and thus variable, they are stabilized or immortalized by selections for variants with cancer-specific autonomy. Owing to these inherent variations cancer karyotypes are heterogeneous within clonal margins. To test this theory we analyzed karyotypes and phenotypes of SV40-infected human, rat and mouse cells developing into neoplastic clones. In all three systems we found (1) preneoplastic aneuploidies, (2) neoplastic clones with individual clonal but flexible karyotypes and phenotypes, which arose from less than one in 10,000 infected cells, survived over 200 generations, but were either T-antigen positive or negative, (3) spontaneous and drug-induced variations of neoplastic phenotypes correlating 1-to-1 with karyotypic variations. Since all 14 virus-induced neoplastic clones tested contained individual clonal karyotypes and

  7. Development of a cell line from Echinococcus granulosus germinal layer.

    PubMed

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  8. Further observations on the behavioral and neural effects of bone marrow stromal cells in rodent pain models

    PubMed Central

    Guo, Wei; Chu, Yu-Xia; Imai, Satoshi; Yang, Jia-Le; Zou, Shiping; Mohammad, Zaid; Wei, Feng; Dubner, Ronald

    2016-01-01

    Background Bone marrow stromal cells (BMSCs) have shown potential to treat chronic pain, although much still needs to be learned about their efficacy and mechanisms of action under different pain conditions. Here, we provide further convergent evidence on the effects of BMSCs in rodent pain models. Results In an orofacial pain model involving injury of a tendon of the masseter muscle, BMSCs attenuated behavioral pain conditions assessed by von Frey filaments and a conditioned place avoidance test in female Sprague-Dawley rats. The antihyperalgesia of BMSCs in females lasted for <8 weeks, which is shorter than that seen in males. To relate preclinical findings to human clinical conditions, we used human BMSCs. Human BMSCs (1.5 M cells, i.v.) attenuated mechanical and thermal hyperalgesia induced by spinal nerve ligation and suppressed spinal nerve ligation-induced aversive behavior, and the effect persisted through the 8-week observation period. In a trigeminal slice preparation, BMSC-treated and nerve-injured C57B/L mice showed reduced amplitude and frequency of spontaneous excitatory postsynaptic currents, as well as excitatory synaptic currents evoked by electrical stimulation of the trigeminal nerve root, suggesting inhibition of trigeminal neuronal hyperexcitability and primary afferent input by BMSCs. Finally, we observed that GluN2A (N-methyl-D-aspartate receptor subunit 2A) tyrosine phosphorylation and protein kinase Cgamma (PKCγ) immunoreactivity in rostral ventromedial medulla was suppressed at 8 weeks after BMSC in tendon-injured rats. Conclusions Collectively, the present work adds convergent evidence supporting the use of BMSCs in pain control. As PKCγ activity related to N-methyl-D-aspartate receptor activation is critical in opioid tolerance, these results help to understand the mechanisms of BMSC-produced long-term antihyperalgesia, which requires opioid receptors in rostral ventromedial medulla and apparently lacks the development of tolerance

  9. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

    PubMed

    Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

    2015-11-01

    Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

  10. Development and characterization of a largemouth bass cell line.

    PubMed

    Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014.

  11. Development and application of human cell lines engineered to metabolically activate structurally diverse environmental mutagens

    NASA Astrophysics Data System (ADS)

    Crespi, C. I.; Langenbach, Robert; Gonzalez, Frank J.; Gelboin, Harry V.; Penman, B. W.

    1993-03-01

    Cytochromes P450 are responsible for the mutagenic/carcinogenic activation of many environmental promutagens/procarcinogens. These enzymes are present at highest concentrations in liver in vivo but are markedly absent in tester organisms for most in vitro mutagenicity test systems. Two approaches have been used to supply needed metabolic activation, incorporation of an extracellular activating system, usually derived from a rodent liver and introduction of activating enzymes into the target cell. The latter approach appears to result in a more sensitive testing system because of the close proximity of the activating enzymes and the target DNA. Human cell lines have been developed which stably express human cytochromes P450 and other cDNAs which have been introduced individually or in combination. The resulting cell lines are exquisitely sensitive to exposure to promutagens and procarcinogens. Mutagenicity is measured at the hypoxanthine phosphoribosyl transferase (hprt) and thymidine kinase (tk) gene loci. The most versatile cell line, designated MCL-5, stably express five cDNAs encoding all of the human hepatic P450s known to be principally responsible for known human procarcinogen activation. The induction of mutation is observed in MCL-5 cells upon exposure to ng/ml levels of model compounds such as nitrosamines, aflatoxin B1 and benzo(a)pyrene. A lower volume mutagenicity assay has been developed for use with samples available in limited amounts. Human lymphoblast mutation assays have been used to screen for mutagenic activity sediment samples from a polluted watershed. Two sediment samples were found to have mutagenic activity to human lymphoblasts.

  12. [Decontamination of continual cell lines spontaneously infected with mycoplasmas].

    PubMed

    Machatková, M; Jurmanová, K; Snejdar, V

    1986-07-01

    The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out.

  13. Radiosensitizing effects of the prenyltransferase inhibitor AZD3409 against RAS mutated cell lines.

    PubMed

    Cengel, Keith A; Deutsch, Eric; Stephens, Trevor C; Voong, K Ranh; Kao, Gary D; Bernhard, Eric J

    2006-09-01

    Mutations at the H-, N- and K-ras loci are among the most frequent genetic alterations in human cancers. In this study, we have investigated the effect of AZD3409, a novel, peptidomimetic prenyltransferase inhibitor (PTI), on the radiosensitivity of cells with mutated ras alleles. AZD3409, developed by AstraZeneca, inhibits both farnesyl- and geranylgeranyl transferase in cell free systems. AZD3409 inhibits the growth of a variety of human cancer cell lines, including cells that express mutant alleles of either K- or H- ras and was well tolerated when administered orally to healthy volunteers in a phase I clinical trial. We have previously shown that PTI can radiosensitize human and rodent cancer cell lines that express activated RAS. Here we assessed the ability of AZD3409 to radiosensitize human cancer cell lines in vivo and in vitro and the activation state of RAS proteins in treated cells. Once daily oral administration of AZD3409 to nude mice bearing PSN-1 and MiaPaCa-2 human pancreatic cancer xenografts expressing mutant K-ras was well tolerated and resulted in a supra-additive reduction in clonogenic cell survival after irradiation. Similarly, AZD3409 reduced clonogenic survival in cells that express either mutant K- or H- ras in vitro. We next examined the effect of AZD3409 on the processing and activation of K- and H-RAS. AZD3409-mediated radiosensitization, both in vivo and in vitro, correlates with a decrease in H-RAS processing without detectable effect on K-RAS processing. RAS activation assays show that the decreased H-RAS processing is accompanied by decreased H-RAS activation in cell lines with mutations in either K- or H-ras. However, no decrease in K-RAS activation was detected. Thus, radiosensitization of human cancer cells that express mutated K-RAS occurred under conditions where AZD3409 inihibits the activation of farneyslated H-RAS, but did not inhibit K-RAS activation.

  14. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    PubMed

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  15. Sensitivity of Hodgkin's lymphoma cell lines to the cell cycle inhibitor roscovitine.

    PubMed

    Foell, Juergen L; Max, Daniela; Giersberg, Corinna; Korholz, Dieter; Staege, Martin S

    2008-01-01

    The prognosis of patients with Hodgkin's lymphoma (HL) has improved in recent decades. However, not all patients can be cured and the development of alternative treatment strategies is necessary. Gene expression in HL cell lines was analyzed using DNA microarrays and both conventional and quantitative reverse transcriptase-polymerase chain reaction. Sensitivity of HL cell lines to the cell cycle inhibitor roscovitine was assessed in vitro. All HL cell lines express high levels of cyclin D2. Treatment of HL cells with roscovitine induced cell death in some cell lines whereas other cell lines were resistant to roscovitine. Roscovitine-sensitive cell lines were characterized by expression of T-cell markers and expressed high levels of the unusual cytokine interleukin-26. Roscovitine is a cytotoxic drug for a subpopulation of HL cells and might be an interesting agent for the treatment of patients with HL.

  16. Bursting by taste-responsive cells in the rodent brain stem

    PubMed Central

    Baird, John-Paul; Tordoff, Michael G.

    2015-01-01

    Neurons that fire in bursts have been well-characterized in vision and other neural systems, but not in taste systems. We therefore examined whether brain stem gustatory neurons fire in bursts during spontaneous activity and, if so, whether such cells differ from nonbursting cells in other characteristics. We looked at neurons in the nucleus of the solitary tract (NST) of C57BL/6ByJ (B6) and 129P3/J (129) mice, and in the NST and parabrachial nucleus (PBN) of Sprague-Dawley rats. Many NST cells fired frequently with short intervals characteristic of bursting, and such neurons differed from others in their responsiveness to taste compounds. In B6 mice and rats, there was a significant positive correlation between the prevalence of short-interval firing and the net spikes evoked by application of NaCl. In contrast, in 129 mice the prevalence of short intervals was positively correlated with the size of sucrose responses. We also compared breadth-of-tuning measures based on counting either all spikes or only those following short intervals, and we found narrower tuning for the latter in the NST of B6 mice and rats. There was little evidence of spontaneous bursting in the rat PBN, and firing patterns in this nucleus were not related to the size of taste-evoked responses. We suggest that bursting may be a strategy employed by the NST to amplify the postsynaptic impact of particular taste stimuli, depending on an animal's needs. Another function may be to sharpen breadth-of-tuning and thus enhance the contrast between stimuli of different taste qualities. PMID:25609109

  17. AZT, rodent somatic and germ cell mutagenicity and reproductive toxicity tests

    SciTech Connect

    Shelby, M.D.; Russell, L.B.; Generoso, W.

    1995-11-01

    AZT (3`-axido-3`-deoxythymidine, Zidovudine) is the most widely used therapeutic agent in the treatment of Acquired Immune Deficiency Syndrome (AIDS). Use of AZT has not been limited to HIV-seropositive individuals or to those with symptoms of AIDS. It has also been used as a chemoprophylactic agent in people accidentally exposed to HIV-contaminated body fluids, and to HIV-seropositive pregnant women to prevent infection of the fetus. Because of these latter uses, it is particularly important to determine whether long-term health effects might be associated with AZT exposure. Tests have been conducted to determine the in vivo genetic toxicity of AZT in mice. Dominant-lethal and morphological-specific-locus tests were conducted in males using 2 daily initraperitoneal injections of 750 mg/kg. The dominant-lethal test was negative for all germ cell stages from differentiating spermatogonia to mature sperm. Likewise, no evidence of the induction of specific locus mutations was observed in either spermatogonial stem cells or poststem-cell stages. Further, tests for effects on male and female reproduction and in utero development indicate a lack of effects. These results, along with preliminary clinical reports that birth outcomes are normal in newborns exposed to AZT in utero, are encouraging with regard to the risks to offspring of parents exposed to AZT, either prior to or during pregnancy. However, positive results in mouse bone marrow micronucleus tests and one report on the induction of chromosomal aberrations in the lymphocytes of AIDS patients on AZT therapy indicate that further studies are needed on the potential of AZT to adversely affect the long-term health of exposed individuals.

  18. Apoptotic effect of noscapine in breast cancer cell lines.

    PubMed

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  19. Interaction of Ionizing Radiation, Genetically Active Chemicals, and Radiofrequency Radiation in Human and Rodent Cells

    DTIC Science & Technology

    1990-12-01

    proflavin , a drug known to intercalate with DNA. Again, when cells were exposed simultaneously to RFR SAR = 40.8- + 13.4 (SD) W/kg or 40 W/kg at power...densities of 87 or 65 mW/cm ), no effect of the RFR on the proflavin induced mutagenicity was observed (Meltz et al., 1990). SCE Induction Previously...Meltz ML, Eagan P, and Erwin DN (1990). Proflavin and Microwave Radiation: Absence of a Mutagenic Interaction. Bioelectromagnetics 11:149-157. Ciaravino

  20. Evaluation of the toxicity of zinc in the rat olfactory neuronal cell line, Odora.

    PubMed

    Hsieh, H; Amlal, H; Genter, M B

    2015-03-01

    Zinc (Zn) has long been touted as a panacea for common cold. Recently, there has been some controversy over whether an intranasal (IN) zinc gluconate gel, purported to fight colds, causes anosmia, or loss of the sense of smell, in humans. Previous evidence has shown that IN zinc sulfate (ZnSO4) solutions can cause anosmia in humans as well as significant damage to the olfactory epithelium in rodents. Using an in vitro olfactory neuron model (the rat Odora cell line), we tested the hypothesis that Zn toxicity was caused by inhibition of the hydrogen voltage-gated channel 1(HVCN1), leading to acidosis and apoptotic cell death. Following studies to characterize the toxicity of zinc gluconate and ZnSO4, Odora cells were grown on coverslips and loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester to measure intracellular pH in the presence and absence of Zn salts. While we found that HVCN1 is not functional in Odora cells, we found that olfactory neurons in vitro maintain their intracellular pH through a sodium/proton exchanger, specifically the sodium proton antiporter 1. ZnSO4, at nontoxic levels, had no impact on intracellular pH after acute exposure or after 24 h of incubation with the cells. In conclusion, Zn toxicity is not mediated through an acidification of intracellular pH in olfactory neurons in vitro. © The Author(s) 2015.

  1. Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

    PubMed Central

    Andersson, Lotta E.; Valtat, Bérengère; Bagge, Annika; Sharoyko, Vladimir V.; Nicholls, David G.; Ravassard, Philippe; Scharfmann, Raphael; Spégel, Peter; Mulder, Hindrik

    2015-01-01

    Aims/Hypothesis Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets. Methods Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets. Results Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells. Conclusions/Interpretation Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings

  2. Expression of a cDNA sequence encoding human purine nucleoside phosphorylase in rodent and human cells.

    PubMed Central

    McIvor, R S; Goddard, J M; Simonsen, C C; Martin, D W

    1985-01-01

    A cDNA sequence which contains the entire coding region for human purine nucleoside phosphorylase (PNP) was recombined for selection and expression in mammalian cells. Plasmids containing either the simian virus 40 early promoter or the mouse metallothionein promoter positioned just upstream of the PNP coding sequence were constructed. These plasmids also contained the gene for a methotrexate-resistant dihydrofolate reductase, allowing for selection and amplification of positive transferrents after transfection of cells by the DNA-calcium phosphate coprecipitation technique. Expression of human PNP activity was readily detected in both mouse (L) and CHO cells by isoelectric focusing of cell extracts followed by histochemical staining for PNP activity. The simian virus 40 early promoter directed considerable expression of human PNP activity in CHO cells but only scant activity in mouse cells. The mouse metallothionein promoter was not successful in effecting human PNP expression in CHO cells but provided substantial human PNP activity in mouse cells and was inducible by incubation with zinc. HeLa cell transferrents were isolated and screened for the presence of transferred PNP cDNA sequences by Southern hybridization analysis. RNA transcripts derived from the transferred PNP cDNA were identified in one of these cell lines. Images PMID:3929070

  3. Disruption of glycosylation enhances ubiquitin-mediated proteasomal degradation of Shadoo in Scrapie-infected rodents and cultured cells.

    PubMed

    Zhang, Jin; Guo, Yan; Xie, Wu-Ling; Xu, Yin; Ren, Ke; Shi, Qi; Zhang, Bao-Yun; Chen, Cao; Tian, Chan; Gao, Chen; Dong, Xiao-Ping

    2014-06-01

    Shadoo (Sho) is an N-glycosylated glycophosphatidylinositol-anchored protein that is expressed in the brain and exhibits neuroprotective properties. Recently, research has shown that a reduction of Sho levels may reflect the presence of PrPSc in the brain. However, the possible mechanism by which prion infection triggers down-regulation of Sho remains unclear. In the present study, Western blot and immunohistochemical assays revealed that Sho, especially glycosylated Sho, declined markedly in the brains of five scrapie agent-infected hamsters and mice at the terminal stages. Analyses of the down-regulation of Sho levels with the emergence of PrPSc C2 proteolytic fragments did not identify close association in all tested scrapie-infected models. To further investigate the mechanism of depletion of Sho in prion disease, a Sho-expressing plasmid with HA tag was introduced into a scrapie-infected cell line, SMB-S15, and its normal cell line, SMB-PS. Western blot assay revealed dramatically decreased Sho in SMB-S15 cells, especially its glycosylated form. Proteasome inhibitor MG132 reversed the decrease of nonglycosylated Sho, but had little effect on glycosylated Sho. N-acetylglucosamine transferase inhibitor tunicamycin efficiently reduced the glycosylations of Sho and PrPC in SMB-PS cells, while two other endoplasmic reticulum stress inducers showed clear inhibition of diglycosylated PrPC, but did not change the expression level and profile of Sho. Furthermore, immunoprecipitation of HA-Sho illustrated ubiquitination of Sho in SMB-S15 cells, but not in SMB-PS cells. We propose that the depletions of Sho in scrapie-infected cell lines due to inhibition of glycosylation mediate protein destabilization and subsequently proteasome degradation after modification by ubiquitination.

  4. FoxP3+ Regulatory T Cells Determine Disease Severity in Rodent Models of Inflammatory Neuropathies

    PubMed Central

    Meyer zu Hörste, Gerd; Cordes, Steffen; Mausberg, Anne K.; Zozulya, Alla L.; Wessig, Carsten; Sparwasser, Tim; Mathys, Christian; Wiendl, Heinz; Hartung, Hans-Peter; Kieseier, Bernd C.

    2014-01-01

    Inflammatory neuropathies represent disabling human autoimmune disorders with considerable disease variability. Animal models provide insights into defined aspects of their disease pathogenesis. Forkhead box P3 (FoxP3)+ regulatory T lymphocytes (Treg) are anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. Dysfunction or a reduced frequency of Tregs have been associated with different human autoimmune disorders. We here analyzed the functional relevance of Tregs in determining disease manifestation and severity in murine models of autoimmune neuropathies. We took advantage of the DEREG mouse system allowing depletion of Treg with high specificity as well as anti-CD25 directed antibodies to deplete Tregs in mice in actively induced experimental autoimmune neuritis (EAN). Furthermore antibody-depletion was performed in an adoptive transfer model of chronic neuritis. Early Treg depletion increased clinical EAN severity both in active and adoptive transfer chronic neuritis. This was accompanied by increased proliferation of myelin specific T cells and histological signs of peripheral nerve inflammation. Late stage Treg depletion after initial disease manifestation however did not exacerbate inflammatory neuropathy symptoms further. We conclude that Tregs determine disease severity in experimental autoimmune neuropathies during the initial priming phase, but have no major disease modifying function after disease manifestation. Potential future therapeutic approaches targeting Tregs should thus be performed early in inflammatory neuropathies. PMID:25286182

  5. Establishment and characterization of a chicken mononuclear cell line.

    PubMed

    Qureshi, M A; Miller, L; Lillehoj, H S; Ficken, M D

    1990-11-01

    A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.

  6. 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.

  7. Investigation of the selenium metabolism in cancer cell lines.

    PubMed

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-02-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 μM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

  8. Establishment of shrimp cell lines: perception and orientation.

    PubMed

    Jayesh, P; Seena, Jose; Singh, I S Bright

    2012-09-01

    Development of continuous shrimp cell lines for effective investigation on shrimp viruses remains elusive with an arduous history of over 25 years. Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. Though improved growth and longevity of shrimp cells in vitro could be achieved by using modified growth media this did not make any leap to spontaneous transformation; probably due to the fact that shrimp cells inhibited neoplastic transformations. Oncogenic induction and immortalization are considered as the possible ways, and an exclusive medium for shrimp cell culture and an appropriate mode of transformation are crucial. In this review status of shrimp cell line development and its future orientation are discussed.

  9. Induction of apoptosis by opium in some tumor cell lines.

    PubMed

    Khaleghi, M; Farsinejad, A; Dabiri, S; Asadikaram, G

    2016-09-30

    The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

  10. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    PubMed

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines.

  11. Retinal Ganglion Cell Damage in an Experimental Rodent Model of Blast-Mediated Traumatic Brain Injury

    PubMed Central

    Mohan, Kabhilan; Kecova, Helga; Hernandez-Merino, Elena; Kardon, Randy H.; Harper, Matthew M.

    2013-01-01

    Purpose. To evaluate retina and optic nerve damage following experimental blast injury. Methods. Healthy adult mice were exposed to an overpressure blast wave using a custom-built blast chamber. The effects of blast exposure on retina and optic nerve function and structure were evaluated using the pattern electroretinogram (pERG), spectral domain optical coherence tomography (OCT), and the chromatic pupil light reflex. Results. Assessment of the pupil response to light demonstrated decreased maximum pupil constriction diameter in blast-injured mice using red light or blue light stimuli 24 hours after injury compared with baseline in the eye exposed to direct blast injury. A decrease in the pupil light reflex was not observed chronically following blast exposure. We observed a biphasic pERG decrease with the acute injury recovering by 24 hours postblast and the chronic injury appearing at 4 months postblast injury. Furthermore, at 3 months following injury, a significant decrease in the retinal nerve fiber layer was observed using OCT compared with controls. Histologic analysis of the retina and optic nerve revealed punctate regions of reduced cellularity in the ganglion cell layer and damage to optic nerves. Additionally, a significant upregulation of proteins associated with oxidative stress was observed acutely following blast exposure compared with control mice. Conclusions. Our study demonstrates that decrements in retinal ganglion cell responses can be detected after blast injury using noninvasive functional and structural tests. These objective responses may serve as surrogate tests for higher CNS functions following traumatic brain injury that are difficult to quantify. PMID:23620426

  12. Natural Killer Cells for Immunotherapy - Advantages of the NK-92 Cell Line over Blood NK Cells.

    PubMed

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

    Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient's blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects.

  13. Guanylate-Binding Protein-1 protects ovarian cancer cell lines but not breast cancer cell lines from killing by paclitaxel.

    PubMed

    Tipton, Aaron R; Nyabuto, Geoffrey O; Trendel, Jill A; Mazur, Travis M; Wilson, John P; Wadi, Suzan; Justinger, Jacob S; Moore, Garret L; Nguyen, Peter T; Vestal, Deborah J

    2016-09-30

    Forced expression of the cytokine-induced large GTPase, human Guanylate-Binding Protein-1 (hGBP-1), in ovarian cancer cell lines increases resistance to paclitaxel. Elevated hGBP-1 RNA in ovarian tumors correlates with shorter recurrence-free survival. In contract, hGBP-1 is part of a gene signature predicting improved prognosis in all subtypes of breast cancers. hGBP-1 does not confer paclitaxel resistance on MCF-7 and TMX2-28 breast cancer cells. Expression of the isotype of the hGBP-1-interacting protein, PIM1, which may contribute to paclitaxel resistance when associated with hGBP-1, is different in breast and ovarian cancer cell lines. Breast cancer cell lines express the 44 kDa isoform of PIM-1, and ovarian cancer cell lines express the 33 kDa isoform. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    SciTech Connect

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  15. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  16. Toxicity of synthetic flavorings, nature identical and artificial, to hematopoietic tissue cells of rodents.

    PubMed

    Sales, I M S; Silva, J M; Moura, E S R; Alves, F D S; Silva, F C C; Sousa, J M C; Peron, A P

    2017-08-17

    The goal of this study was to analyze cytotoxicity, genotoxicity and mutagenicity to bone marrow cells of mice of nature identical synthetic flavorings, passion fruit and strawberry, and artificial synthetic flavorings, vanilla, chocolate, tutti-frutti and cookie, at doses 0.5; 1.0; 2.0; 5.0 and 10.0 mL/kg. The additives were given to the animals by gavage in a single daily application for seven days. Data were subjected to analysis of variance (ANOVA) followed by post Tukey's post hoc test, p <0.05. Animals treated with 2.0; 5.0 and 10.0 mL/Kg of flavorings chocolate, strawberry and cookie, and 5.0 and 10.0 mL/Kg of flavorings vanilla and passion fruit died on the fifth and sixth day of the experiment, respectively. The doses 0.5 and 1.0 mL/Kg of the six additives significantly reduced erythropoiesis in the examined tissue. Also, treatments 0.5 and 1.0 mL/Kg of chocolate, and 1.0 mL/Kg of strawberry and biscuit induced the formation of micronuclei in the bone marrow erythrocytes, at a significant frequency. Therefore, under the study conditions, the six microingredients analyzed were cytotoxic and genotoxic, and additives strawberry, chocolate and cookie were also mutagenic in at least one of the evaluated doses.

  17. Knockout of Ccr2 alleviates photoreceptor cell death in rodent retina exposed to chronic blue light.

    PubMed

    Hu, Zizhong; Zhang, Yi; Wang, Junling; Mao, Pingan; Lv, Xuehua; Yuan, Songtao; Huang, Zhengru; Ding, Yuzhi; Xie, Ping; Liu, Qinghuai

    2016-11-10

    Age-related macular degeneration (AMD), the leading cause of visual loss after the age of 60 years, is a degenerative retinal disease involving a variety of environmental and hereditary factors. Although it has been implicated that immune system is involved in the disease progression, the exact role that microglia has is still unclear. Here we demonstrated that knockout of Ccr2 gene could alleviate photoreceptor cell death in mice retinas exposed to chronic blue light. In Ccr2(-/-) mice, a damaged microglia recruitment was shown in retina and this could protect the visual function in electroretinogram and alleviate the photoreceptor apoptosis, which thus helped attenuate the blue light-induced retinopathy. We further found an increased co-location of NLRP3, Iba-1, and IL-1β in fluorescence and a concomitant increased protein expression of NLRP3, caspase-1, and IL-1β in western blotting in chronic blue light-induced retinopathy. Moreover, the activation of microglia and their cellular NLRP3 inflammasomes occurred as an earlier step before the structural and functional damage of the mice retinas, which collectively supported that microglial NLRP3 inflammasome might be the key to the chronic blue light-induced retinopathy.

  18. Reliable in vitro studies require appropriate ovarian cancer cell lines

    PubMed Central

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  19. Efficacy and dose-dependent safety of intra-arterial delivery of mesenchymal stem cells in a rodent stroke model.

    PubMed

    Yavagal, Dileep R; Lin, Baowan; Raval, Ami P; Garza, Philip S; Dong, Chuanhui; Zhao, Weizhao; Rangel, Erika B; McNiece, Ian; Rundek, Tatjana; Sacco, Ralph L; Perez-Pinzon, Miguel; Hare, Joshua M

    2014-01-01

    Intra-arterial (IA) delivery of mesenchymal stem cells (MSCs) for acute ischemic stroke is attractive for clinical translation. However, studies using rat model of stroke have demonstrated that IA MSCs delivery can decrease middle cerebral artery (MCA) flow, which may limit its clinical translation. The goal of this study is to identify a dose of IA MSCs (maximum tolerated dose; MTD) that does not compromise MCA flow and evaluate its efficacy and optimal timing in a rat model of reversible middle cerebral artery occlusion (rMCAo). We sought to determine if there is a difference in efficacy of acute (1 h) versus sub-acute (24 h) IA MSCs treatment after rMCAo. Adult female Sprague-Dawley rats underwent rMCAo (90 min) and an hour later a single dose of MSCs (at de-escalating doses 1 × 10(6), 5 × 10(5), 2 × 10(5), 1 × 10(5) and 5 × 10(4)) was given using IA route. MSCs were suspended in phosphate buffered saline (PBS) and PBS alone was used for control experiments. We measured the percent change in mean laser Doppler flow signal over the ipsilateral MCA in de-escalating doses groups to determine MTD. The results demonstrated that the lowering of IA MSC dose to 1 × 10(5) and below did not compromise MCA flow and hence an IA MSC dose of 1 × 10(5) considered as MTD. Subsequently, 1 h and 24 h after rMCAo, rats were treated with IA MSCs or PBS. The 24 h delivery of IA MSCs significantly improved neurodeficit score and reduced the mean infarct volume at one month as compared to control, but not the 1 h delivery. Overall, this study suggests that the IA delivery of MSCs can be performed safely and efficaciously at the MTD of 1 × 10(5) delivered at 24 hours in rodent model of stroke.

  20. Inhibition of DMBA-induced Oral Squamous Cells Carcinoma Growth by Brazilian Red Propolis in Rodent Model.

    PubMed

    Ribeiro, Danielle R; Alves, Ângela Valéria F; dos Santos, Esaú P; Padilha, Francine F; Gomes, Margarete Z; Rabelo, Alessandra S; Cardoso, Juliana C; Massarioli, Adna Prado; de Alencar, Severino Matias; de Albuquerque-Júnior, Ricardo Luiz C

    2015-08-01

    We investigated the effect of oral administration of hydroalcoholic extract of Brazilian red propolis (HERP) on DMBA-induced oral squamous cell carcinomas (OSCC) in rodents. The chemical components of the HERP were assessed by high-performance liquid chromatography (HPLC). Carcinogenesis was topically induced in the lower lip of 25 rats using 9,10-dimethyl-1,2-benzanthracene (DMBA); the tumour was treated with saline (TUM1) and Tween 80 (TUM2) as well as HERP at 10, 50 and 100 mg/kg (HERP10, HERP50 and HERP100, respectively) for 20 weeks. Topical application of saline and oral administration of 100 mg/kg HERP was used in five rats as a control group (CTR). After 26 weeks, the histological malignancy grading and immunohistochemical expression of Ki-67 and p16(INK4A) were assessed in the tumours/tissue samples. The compounds identified were propyl gallate, daidzein, catechin, epicatechin, formononetin and biochanin A. Formononetin, daidzein and biochanin A showed concentration of 23.29, 0.38 and 0.67 mg/g of HERP, respectively. HERP at doses of 50 and 100 mg/kg inhibited 40% of OSCC growth and promoted a 3-week delay in development of clinically detectable tumours. Epithelial dysplasia was observed in all samples with no clinical tumour, except in CTR. No significant difference in the immunoexpression of Ki-67 and p16(INK4A) was observed between HERP-treated and saline/Tween 80-treated groups (p > 0.05). Our results suggest that HERP exerts chemopreventive activity on the progression of DMBA-induced epithelial dysplasia to OSCC in an experimental model of labial carcinogenesis; however, this effect is not associated with Ki-67 and p16(INK4A) immunoexpression. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  1. Mammalian cell line developments in speed and efficiency.

    PubMed

    Estes, Scott; Melville, Mark

    2014-01-01

    Mammalian cell expression systems are the dominant tool today for producing complex biotherapeutic proteins. In this chapter, we discuss the basis for this dominance, and further explore why the Chinese hamster ovary (CHO) cell line has become the prevalent choice of hosts to produce most recombinant biologics. Furthermore, we explore some of the innovations that are currently in development to improve the CHO cell platform, from cell line specific technologies to overarching technologies that are designed to improve the overall workflow of bioprocess development.

  2. Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines

    PubMed Central

    Gabrani, Reema; Jain, Ramya; Sharma, Anjali; Sarethy, Indira P.; Dang, Shweta; Gupta, S.

    2012-01-01

    Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines. PMID:23716874

  3. Kinetic and pharmacological properties of the M-current in rodent neuroblastoma x glioma hybrid cells.

    PubMed

    Robbins, J; Trouslard, J; Marsh, S J; Brown, D A

    1992-01-01

    1. The M-like current IK(M,ng) in differentiated NG108-15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight-seal, whole-cell patch-clamp recording. 2. When calculated from steady-state current-voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half-activation voltage Vo = -44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e-fold increase in conductance. In 12 mM [K+] Vo = -38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e-fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady-state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo-1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)-free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9-amino-1,2,3,4-tetrahydroacridine

  4. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines.

    PubMed

    Harenza, Jo Lynne; Diamond, Maura A; Adams, Rebecca N; Song, Michael M; Davidson, Heather L; Hart, Lori S; Dent, Maiah H; Fortina, Paolo; Reynolds, C Patrick; Maris, John M

    2017-03-28

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma.

  5. Baculovirus studies in new, indigenous lepidopteran cell lines.

    PubMed

    Pant, U; Sudeep, A B; Athawale, S S; Vipat, V C

    2002-01-01

    Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines.

  6. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines

    PubMed Central

    Harenza, Jo Lynne; Diamond, Maura A.; Adams, Rebecca N.; Song, Michael M.; Davidson, Heather L.; Hart, Lori S.; Dent, Maiah H.; Fortina, Paolo; Reynolds, C. Patrick; Maris, John M.

    2017-01-01

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma. PMID:28350380

  7. Establishment of the DU.528 human lymphohemopoietic stem cell line

    PubMed Central

    1985-01-01

    We have established the DU.528 cell line from the pretreatment leukemia cells of a patient who underwent a T lymphoblastic-to-promyelocytic phenotype conversion during treatment with the adenosine deaminase inhibitor, deoxycoformycin. The cell line and clones obtained from it by limiting dilution have the same karyotype previously found in the patient's pretreatment T lymphoblasts and post-deoxycoformycin treatment promyelocytes. DU.528 cells in continuous culture for greater than 2 yr display a predominant undifferentiated T lymphoblastoid phenotype. These cells spontaneously generate progeny of at least three lineages, T lymphoid, granulocytic/monocytic, and erythroid. The surface marker most consistently expressed by DU.528 cells in the undifferentiated state is the 3A1 antigen, which has been found on prothymocytes in the embryonic thymus. Some undifferentiated DU.528 cells also expressed the IL-2 receptor, but no other T cell differentiation antigens. Exposure of DU.528 cells to a variety of agents induced myeloid maturation; adenosine and deoxyadenosine, in the presence of deoxycoformycin, induced expression of myeloid differentiation antigens. Our results suggest that DU.528 is a lymphohematopoietic stem cell line and support the hypothesis that differentiation of pluripotent stem cells may be altered by genetic deficiency of adenosine deaminase. DU.528 cells may provide a useful model for examining factors that regulate stem cell proliferation and differentiation. PMID:4056659

  8. Picking Cell Lines for High-Throughput Transcriptomic Toxicity ...

    EPA Pesticide Factsheets

    High throughput, whole genome transcriptomic profiling is a promising approach to comprehensively evaluate chemicals for potential biological effects. To be useful for in vitro toxicity screening, gene expression must be quantified in a set of representative cell types that captures the diversity of potential responses across chemicals. The ideal dataset to select these cell types would consist of hundreds of cell types treated with thousands of chemicals, but does not yet exist. However, basal gene expression data may be useful as a surrogate for representing the relevant biological space necessary for cell type selection. The goal of this study was to identify a small (< 20) number of cell types that capture a large, quantifiable fraction of basal gene expression diversity. Three publicly available collections of Affymetrix U133+2.0 cellular gene expression data were used: 1) 59 cell lines from the NCI60 set; 2) 303 primary cell types from the Mabbott et al (2013) expression atlas; and 3) 1036 cell lines from the Cancer Cell Line Encyclopedia. The data were RMA normalized, log-transformed, and the probe sets mapped to HUGO gene identifiers. The results showed that <20 cell lines capture only a small fraction of the total diversity in basal gene expression when evaluated using either the entire set of 20960 HUGO genes or a subset of druggable genes likely to be chemical targets. The fraction of the total gene expression variation explained was consistent when

  9. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    PubMed

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test.

  10. Inducible human immunodeficiency virus type 1 packaging cell lines.

    PubMed Central

    Yu, H; Rabson, A B; Kaul, M; Ron, Y; Dougherty, J P

    1996-01-01

    Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. PMID:8676479

  11. Establishment and characterization of a novel osteosarcoma cell line: CHOS.

    PubMed

    Liu, Yunlu; Feng, Xiaobo; Zhang, Yukun; Jiang, Hongyan; Cai, Xianyi; Yan, Xinxin; Huang, Zengfa; Mo, Fengbo; Yang, Wen; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

    2016-12-01

    Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  12. Drug/Cell-line Browser: interactive canvas visualization of cancer drug/cell-line viability assay datasets.

    PubMed

    Duan, Qiaonan; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Benes, Cyril H; Ma'ayan, Avi

    2014-11-15

    Recently, several high profile studies collected cell viability data from panels of cancer cell lines treated with many drugs applied at different concentrations. Such drug sensitivity data for cancer cell lines provide suggestive treatments for different types and subtypes of cancer. Visualization of these datasets can reveal patterns that may not be obvious by examining the data without such efforts. Here we introduce Drug/Cell-line Browser (DCB), an online interactive HTML5 data visualization tool for interacting with three of the recently published datasets of cancer cell lines/drug-viability studies. DCB uses clustering and canvas visualization of the drugs and the cell lines, as well as a bar graph that summarizes drug effectiveness for the tissue of origin or the cancer subtypes for single or multiple drugs. DCB can help in understanding drug response patterns and prioritizing drug/cancer cell line interactions by tissue of origin or cancer subtype. DCB is an open source Web-based tool that is freely available at: http://www.maayanlab.net/LINCS/DCB CONTACT: avi.maayan@mssm.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. The Type 1 Alveolar Lining Cells of the Mammalian Lung

    PubMed Central

    Rosenbaum, Robert M.; Picciano, Paul

    1978-01-01

    Using a newly described dissociation and isolation technique, Type 1 alveolar lining cells were obtained from adult rabbit lung within a heterogeneous population. Identification of many lung cell types in this mixed population was by a) comparison of isolated cells with in situ lung cells in lung sections using identical parallel staining, b) stepwise ultrastructural examination of cells during all stages of lung dissociation so that intercellular associations were monitored throughout, and c) Type 1 cell surface changes following collagenase treatment. This phenomenon was studied with both electron and light microscopy, the latter employing tetrachrome staining of basophilic blebs as well as characteristic staining of nucleus and cytoplasm. Following their isolation, most Type 1 cells lost their surface blebs and assumed a “relaxed” state. In this condition, Type 1 cells were exposed to cytochalasin D (CD) for various times and at several concentrations. Surface knobs, having all the characteristics of zeiotic knobs produced in a number of cultured cell lines by exposure to CD, were produced in isolated Type 1 epithelial cells within 45 minutes. The reaction to CD was temperature-dependent, proceeding maximally at 37 C with inhibition at lower temperatures and was inhibited by antimetabolites such as dinitrophenol and 2-deoxyglucose in the presence of CD. As with established cell lines, formation of zeiotic knobs at the isolated Type 1 cell surface appeared closely related to microfilamentous nets located beneath the plasmalemma. The density of this net appeared to vary as isolated Type 1 cells underwent expansion and contraction in response to CD. Zeiotic knobs were formed as the result of herniation of endoplasm through the cell cortex. The significance of such a labile cortical zone is considered in relation to the deformation changes Type 1 cells undergo during inflation-deflation of alveoli and the folding-unfolding of alveolar lining cells as a result of

  14. On-line characterization of a hybridoma cell culture process.

    PubMed

    Zhou, W; Hu, W S

    1994-06-20

    The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.

  15. Antitumor Effects of Vitamin D Analogs on Hamster and Mouse Melanoma Cell Lines in Relation to Melanin Pigmentation

    PubMed Central

    Wasiewicz, Tomasz; Szyszka, Paulina; Cichorek, Miroslawa; Janjetovic, Zorica; Tuckey, Robert C.; Slominski, Andrzej T.; Zmijewski, Michal A.

    2015-01-01

    Deregulated melanogenesis is involved in melanomagenesis and melanoma progression and resistance to therapy. Vitamin D analogs have anti-melanoma activity. While the hypercalcaemic effect of the active form of Vitamin D (1,25(OH)2D3) limits its therapeutic use, novel Vitamin D analogs with a modified side chain demonstrate low calcaemic activity. We therefore examined the effect of secosteroidal analogs, both classic (1,25(OH)2D3 and 25(OH)D3), and novel relatively non-calcemic ones (20(OH)D3, calcipotriol, 21(OH)pD, pD and 20(OH)pL), on proliferation, colony formation in monolayer and soft-agar, and mRNA and protein expression by melanoma cells. Murine B16-F10 and hamster Bomirski Ab cell lines were shown to be effective models to study how melanogenesis affects anti-melanoma treatment. Novel Vitamin D analogs with a short side-chain and lumisterol-like 20(OH)pL efficiently inhibited rodent melanoma growth. Moderate pigmentation sensitized rodent melanoma cells towards Vitamin D analogs, and altered expression of key genes involved in Vitamin D signaling, which was opposite to the effect on heavily pigmented cells. Interestingly, melanogenesis inhibited ligand-induced Vitamin D receptor translocation and ligand-induced expression of VDR and CYP24A1 genes. These findings indicate that melanogenesis can affect the anti-melanoma activity of Vitamin D analogs in a complex manner. PMID:25811927

  16. Exometabolom analysis of breast cancer cell lines: Metabolic signature

    PubMed Central

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-01-01

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach. PMID:26293811

  17. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

    Guerrero, Carlos A.; Guerrero, Rafael A.; Silva, Elver; Acosta, Orlando; Barreto, Emiliano

    2016-01-01

    A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death. PMID:26828934

  18. Myelination in coculture of established neuronal and Schwann cell lines.

    PubMed

    Sango, Kazunori; Kawakami, Emiko; Yanagisawa, Hiroko; Takaku, Shizuka; Tsukamoto, Masami; Utsunomiya, Kazunori; Watabe, Kazuhiko

    2012-06-01

    Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 μg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 μg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.

  19. Rodents And Other Gnawers.

    ERIC Educational Resources Information Center

    Naturescope, 1986

    1986-01-01

    Presents information about rodents and lagomorphs, including definitions and the characteristics of these animals. Contains teaching activities such as "Habitats for Hoppers,""Cartoon Gnawers," and "The Great Rodent Expedition." Reproducible handouts for two of the activities are provided. (TW)

  20. Sclerostin Antibody Administration Converts Bone Lining Cells Into Active Osteoblasts.

    PubMed

    Kim, Sang Wan; Lu, Yanhui; Williams, Elizabeth A; Lai, Forest; Lee, Ji Yeon; Enishi, Tetsuya; Balani, Deepak H; Ominsky, Michael S; Ke, Hua Zhu; Kronenberg, Henry M; Wein, Marc N

    2016-11-14

    Sclerostin antibody (Scl-Ab) increases osteoblast activity, in part through increasing modeling-based bone formation on previously quiescent surfaces. Histomorphometric studies have suggested that this might occur through conversion of bone lining cells into active osteoblasts. However, direct data demonstrating Scl-Ab-induced conversion of lining cells into active osteoblasts are lacking. Here, we used in vivo lineage tracing to determine if Scl-Ab promotes the conversion of lining cells into osteoblasts on periosteal and endocortical bone surfaces in mice. Two independent, tamoxifen-inducible lineage-tracing strategies were used to label mature osteoblasts and their progeny using the DMP1 and osteocalcin promoters. After a prolonged "chase" period, the majority of labeled cells on bone surfaces assumed a thin, quiescent morphology. Then, mice were treated with either vehicle or Scl-Ab (25 mg/kg) twice over the course of the subsequent week. After euthanization, marked cells were enumerated, their thickness quantified, and proliferation and apoptosis examined. Scl-Ab led to a significant increase in the average thickness of labeled cells on periosteal and endocortical bone surfaces, consistent with osteoblast activation. Scl-Ab did not induce proliferation of labeled cells, and Scl-Ab did not regulate apoptosis of labeled cells. Therefore, direct reactivation of quiescent bone lining cells contributes to the acute increase in osteoblast numbers after Scl-Ab treatment in mice. © 2017 American Society for Bone and Mineral Research.

  1. 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

  2. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Marley, Garry M. (Inventor)

    1991-01-01

    Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

  3. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to

  4. Hedgehog signaling pathway is inactive in colorectal cancer cell lines.

    PubMed

    Chatel, Guillaume; Ganeff, Corine; Boussif, Naima; Delacroix, Laurence; Briquet, Alexandra; Nolens, Gregory; Winkler, Rosita

    2007-12-15

    The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines.

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

    PubMed

    Motoyama, T; Hojo, H; Watanabe, H

    1986-01-01

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

  6. Rabeprazole exhibits antiproliferative effects on human gastric cancer cell lines

    PubMed Central

    GU, MENGLI; ZHANG, YAN; ZHOU, XINXIN; MA, HAN; YAO, HANGPING; JI, FENG

    2014-01-01

    Intracellular proton extrusion in gastric cancer cells has been reported to promote cancer cell survival under acidic conditions via hydrogen/potassium adenosine triphosphatase (H+/K+-ATPase). Rabeprazole is a frequently used second-generation proton pump inhibitor (PPI) that irreversibly inactivates gastric H+/K+-ATPase. Therefore, we hypothesized that rabeprazole could reduce the viability of gastric cancer cells. In the present study, four human gastric cancer cell lines and one non-cancer gastric cell line were cultured. Cell viability, the α- and β-subunits of H+/K+-ATPase and cellular apoptosis were analyzed by dye exclusion assay, reverse transcription-polymerase chain reaction and annexin V-fluorescein isothiocyanate/propidium iodide staining, respectively. The expression level of total extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and phosphorylated-ERK protein was detected by western blot analysis. Gastric cancer cell lines were more tolerant of the acidic culture media than non-cancer cells. Administration of rabeprazole led to a marked decrease in the viability of MKN-28 cells. Exposure to rabeprazole induced significant apoptosis in AGS cells. Rabeprazole completely inhibited the phosphorylation of ERK 1/2 in the MKN-28 cells, whereas the same effect was not observed in either the KATO III or MKN-45 cells. The ERK 1/2 inhibitor, PD98059, attenuated the viability of the AGS cells. A similar antiproliferative effect was observed in the rabeprazole treatment group. In addition, PD98059 and rabeprazole were able to efficaciously inhibit the phosphorylation of ERK 1/2 in the gastric cancer cells. Therefore, it was concluded that rabeprazole can attenuate the cell viability of human gastric cancer cells through inactivation of the ERK1/2 signaling pathway. The results of the present study demonstrate that rabeprazole inhibits the viability of gastric cancer cells in vitro and may serve as a novel antineoplastic agent. PMID:25202402

  7. Canine mammary tumour cell lines established in vitro.

    PubMed

    Hellmén, E

    1993-01-01

    Mammary tumours are the most common tumours in the female dog. The tumours have a complex histology and exist in epithelial, mixed and mesenchymal forms. To study the biology of canine mammary tumours, five cell lines have been established and characterized. The results indicate that canine mammary tumours might be derived from mammary stem cells and that the tumour growth is independent of oestrogens. The established canine mammary tumour cell lines will be valuable tools in further studies of the histogenesis and pathogenesis of these tumours.

  8. Definitive Molecular Cytogenetic Characterization of 15 Colorectal Cancer Cell Lines

    PubMed Central

    Knutsen, Turid; Padilla-Nash, Hesed M.; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J.; Ried, Thomas

    2009-01-01

    In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. We here present the results of a comprehensive investigation of 15 established colorectal cancer cell lines utilizing spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) are described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines, isochromosomes were the most common recurrent abnormalities, and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities result predominantly in copy number changes rather than specific chromosome or gene fusions, suggests this may be the major mechanism leading to carcinogenesis in colorectal cancer. PMID:19927377

  9. Definitive molecular cytogenetic characterization of 15 colorectal cancer cell lines.

    PubMed

    Knutsen, Turid; Padilla-Nash, Hesed M; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J; Ried, Thomas

    2010-03-01

    In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. Here, we present the results of a comprehensive investigation of 15 established colorectal cancer cell lines using spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) is described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines; isochromosomes were the most common recurrent abnormalities; and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities predominantly result in copy number changes rather than specific chromosome or gene fusions suggests that this may be the major mechanism leading to carcinogenesis in colorectal cancer.

  10. Guidelines for the use of cell lines in biomedical research.

    PubMed

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-09

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

  11. Design of tunable microwave transmission lines using metamaterial cells

    NASA Astrophysics Data System (ADS)

    Bensafieddine, D.; Djerfaf, F.; Chouireb, F.; Vincent, D.

    2017-04-01

    In this paper, frequency tunable transmission lines are designed using metasurface split ring resonator unit cell. We prove that the tuning principle in metasurface transmission lines is based on the variation of the resonance frequency of the permeability. The frequency-tuning arises by changing the values of two gaps in the inner and outer rings of unit cell ( g1 and g2). The branches of a disconnected gaps type conductor of each unit cell can be joined by switches (PIN diodes, MEMs, etc.). According to switch states ON or OFF, the unit cell has four different commutable behaviors which are 00, 01, 11, and 10. The results show that the resonance frequency of our metasurface transmission line is strongly shifted by about 2.5 GHz between the cases (01) and (11).

  12. Guidelines for the use of cell lines in biomedical research

    PubMed Central

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-01-01

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

  13. Centrosomal proteins and lactate dehydrogenase possess a common epitope in human cell lines.

    PubMed Central

    Gosti, F; Marty, M C; Courvalin, J C; Maunoury, R; Bornens, M

    1987-01-01

    A spontaneously arising rabbit anti-centrosome serum with strong human specificity, used to identify specific antigens in isolated centrosomes, was shown to react with several noncentrosomal proteins including a 36-kDa protein that appeared to be the major cellular antigen. To explore the immunological relationship between noncentrosomal and centrosomal antigens, immunoglobulins were affinity purified using the individual noncentrosomal antigens (from lymphoblastoma KE37 cells) and were tested for their capacity to bind to human centrosomes in situ and to proteins from isolated centrosomes. In this way, the 36-kDa antigen, an abundant cytosolic protein, was shown to share at least one antigenic determinant with high molecular weight centrosomal proteins. This antigen was further identified by mild proteolysis as the glycolytic enzyme lactate dehydrogenase. In all the analyzed human cell lines, the centrosomal staining in situ was correlated with a strong labeling of purified lactate dehydrogenase in immunoblots. Conversely, the absence of centrosomal staining in rodent cells was always correlated with the absence of lactate dehydrogenase labeling. These data suggest an evolutionary relationship between centrosomal proteins and this "housekeeping" enzyme. Images PMID:2434947

  14. Susceptibility of nonprimate cell lines to hepatitis A virus infection.

    PubMed Central

    Dotzauer, A; Feinstone, S M; Kaplan, G

    1994-01-01

    Hepatitis A virus (HAV) has been adapted to grow in primate cell cultures. We investigated replication of HAV in nonprimate cells by inoculating 20 cell lines from different species with the tissue culture-adapted HM175 strain. Slot blot hybridization and immunofluorescence analysis revealed that HAV replicated in GPE, SP 1K, and IB-RS-2 D10 cells of guinea pig, dolphin, and pig origin, respectively. Studies in IB-RS-2 D10 cells were discontinued because cultures were contaminated with classical swine fever virus. A growth curve showed that HAV grew poorly in GPE cells and intermediately in SP 1K cells compared with growth in FRhK-4 cells. Therefore, the cell surface receptor(s) and other host factor(s) required for HAV replication are present in nonprimate as well as primate cells. Images PMID:8057483

  15. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

    Radiation effects, such as bystander effects, hyper radiosensitivity/induced radioresistance (HRS/IRR) and adaptive response that are not related to direct DNA damage are now accepted. However the inter-relationship between them and the possible impact on the scientific basis for radiation protection are highly controversial. This thesis attempts to elucidate the mechanisms of some of these well known but little understood effects. Each paper examines some aspect of bystander effects, adaptive responses and HRS/IRR in an effort to understand how they vary with cell type, dose and time of exposure to single or multiple doses. All the effects involve non-linear dose effect curves and are mainly evident following low doses. Overall findings of the thesis include (1) A clear difference was observed between radioresistant, tumorigenic cell lines with mutant p53 gene expression, and radiosensitive, more normal, cell lines with wild type p53. In general death inducing bystander responses are induced in normal cell populations exposed to low doses of radiation while survival inducing IRR and adaptive responses are seen in the radioresistant tumorigenic cell lines. (2) A cohort of fish cell lines which demonstrated survival promoting bystander effects, also did not show a protective adaptive responses. (3) Adaptive responses traditionally occur when a large challenge dose is given 4--6hrs following low (10--100mGy) priming doses but this thesis shows that for the epithelial cell lines tested, the size of the priming dose (range 0.1--2Gy) does not appear to alter the size of the recovery response. Additionally increased survival could be detected in some cases when the challenge dose was given within one hour of the priming dose. The overall conclusion is that cell lines induce either a bystander response or a protective/adaptive response depending on genetic background and other factors. Care is needed in the interpretation of data generated from only one or two cell lines

  16. Identification and pharmacological characterization of native, functional human urotensin-II receptors in rhabdomyosarcoma cell lines

    PubMed Central

    Douglas, Stephen A; Naselsky, Diane; Ao, Zhaohui; Disa, Jyoti; Herold, Christopher L; Lynch, Frank; Aiyar, Nambi V

    2004-01-01

    In an effort to identify endogenous, native mammalian urotensin-II (U-II) receptors (UT), a diverse range of human, primate and rodent cell lines (49 in total) were screened for the presence of detectable [125I]hU-II binding sites. UT mRNA (Northern blot, PCR) and protein (immunocytochemistry) were evident in human skeletal muscle tissue and cells. [125I]hU-II bound to a homogenous population of high-affinity, saturable (Kd 67.0±11.8 pM, Bmax 9687±843 sites cell−1) receptors in the skeletal muscle (rhabdomyosarcoma) cell line SJRH30. Radiolabel was characteristically slow to dissociate (⩽15% dissociation 90 min). A lower density of high-affinity U-II binding sites was also evident in the rhabdomyosarcoma cell line TE671 (1667±165 sites cell−1, Kd 74±8 pM). Consistent with the profile recorded in human recombinant UT-HEK293 cells, [125I]hU-II binding to SJRH30 cells was selectively displaced by both mammalian and fish U-II isopeptides (Kis 0.5±0.1–1.2±0.3 nM) and related analogues (hU-II[4-11]>[Cys5,10]Acm hU-II; Kis 0.4±0.1 and 864±193 nM, respectively). U-II receptor activation was functionally coupled to phospholipase C-mediated [Ca2+]i mobilization (EC50 6.9±2.2 nM) in SJRH30 cells. The present study is the first to identify the presence of ‘endogenous' U-II receptors in SJRH30 and TE671 cells. SJRH30 cells, in particular, might prove to be of utility for (a) investigating the pharmacological properties of hU-II and related small molecule antagonists at native human UT and (b) delineating the role of this neuropeptide in the (patho)physiological regulation of mammalian neuromuscular function. PMID:15210573

  17. Induction of experimental autoimmune uveoretinitis by T-cell lines.

    PubMed Central

    Rozenszajn, L A; Muellenberg-Coulombre, C; Gery, I; el-Saied, M; Kuwabara, T; Mochizuki, M; Lando, Z; Nussenblatt, R B

    1986-01-01

    Experimental autoimmune uveoretinitis was induced in genetically susceptible Lewis rats by passive transfer of T-lymphocyte cell lines from long-term cultures primed against soluble retinal antigen (S-Ag). A continuous T-cell line was established from non-adherent lymph node cells of S-Ag-immunized Lewis rats. The lymphoid cells were propagated in vitro by serially restimulating them with S-Ag in the presence of irradiated syngeneic spleen cells and expanding them in IL-2-containing media. The cell lines exhibited markers specific for T lymphocytes and the majority had the helper phenotype. When naïve rats were inoculated intravenously with anti S-Ag T-cell lines re-exposed to the antigen prior to injection, they developed uveoretinitis with both clinical and histological characteristics in half the time required by S-Ag to induce the disease by active immunization. The rats exhibited a delayed hypersensitivity skin reaction towards S-Ag. Images Figure 2 Figure 3 PMID:3485569

  18. Cytogenetic characteristics of cell lines from Ixodes scapularis (Acari: Ixodidae).

    PubMed

    Chen, C; Munderloh, U G; Kurtti, T J

    1994-05-01

    Three new cell lines, IDE8 and IDE12 from embryos of northern specimens of Ixodes scapularis Say and ISE18 from southern specimens of I. scapularis, were compared cytogenetically via conventional karyotyping, C- and G-banding, and nucleolar organizing regions (NORs). The karyotypes were very similar. The standard karyotype in the three cell lines consisted of 28 chromosomes with 26 autosomes and XX (female) or XY (male) sex chromosomes. The X chromosome was the largest, and the Y chromosome the smallest chromosome of the karyotype. Constitutive heterochromatin (C-bands) was almost entirely restricted to the centromeric region. An additional interstitial C-band in chromosome 7 was an important notable characteristic of the three cell lines. In sets showing a similar degree of condensation, individual chromosomes of the three lines had identical G-banding patterns. In addition, there was no difference among the cells in number and position of NORs. There were approximately 100 G-bands per haploid set in chromosomes from cells in metaphase, with three to 18 G-bands in each chromosome arm. After staining with silver nitrate, interstitial NORs were identified in chromosomes 7, 10, and the X chromosome. Male cells had five and female cells had six NORs. These findings support the notion that I. scapularis and I. dammini Spielman et al. are conspecific.

  19. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

  20. Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

    DTIC Science & Technology

    2014-06-30

    modified targets in lysed human hepatocyte- like cells (HepaRG) using a direct liquid chromatography−mass spectrometry (LC−MS) assay of cell lysates...parasympathetic autonomic nervous system5 and the neuromuscular systems.3 Recent studies also suggest that DDVP affects non-neuronal targets in human ... human hepatocyte-like cell line (HepaRG) with DDVP. Then, we identified DDVP-modified targets in these lysates either with shotgun proteomics or with a

  1. Butyrate-Induced Apoptosis in Prostate Cancer Cell Lines

    DTIC Science & Technology

    2001-09-01

    butyrate-induced apoptosis was independent of cell cycle phase. 14. SUBJECT TERMS 15. NUMBER OF PAGES prostate cancer, histone deacetylase inhibitors, bone...of cells plated) HDI histone deacetylase inhibitor SBHA suberoylbishydroxamate PKC protein kinase C activator SDS-PAGE SDS polyacrylamide gel...cancer cell lines 1. Summary of goals and findings Histone deacetylase inhibitors (HDI) such as butyrate and suberoylbishydroxamate (SBHA) have

  2. Biological behaviors and proteomics analysis of hybrid cell line EAhy926 and its parent cell line A549.

    PubMed

    Lu, Ze Jun; Ren, Ya Qiong; Wang, Guo Ping; Song, Qi; Li, Mei; Jiang, Sa Sa; Ning, Tao; Guan, Yong Song; Yang, Jin Liang; Luo, Feng

    2009-02-13

    It is well established that cancer cells can fuse with endothelial cells to form hybrid cells spontaneously, which facilitates cancer cells traversing the endothelial barrier to form metastases. However, up to now, little is known about the biologic characteristics of hybrid cells. Therefore, we investigate the malignant biologic behaviors and proteins expression of the hybrid cell line EAhy926 with its parent cell line A549. Cell counting and flow cytometry assay were carried out to assess cell proliferation. The number of cells attached to the extracellular matrix (Matrigel) was measured by MTT assay for the adhesion ability of cells. Transwell chambers were established for detecting the ability of cell migration and invasion. Tumor xenograft test was carried out to observe tumorigenesis of the cell lines. In addition, two-dimensional electrophoresis (2-DE) and mass spectrometry were utilized to identify differentially expressed proteins between in Eahy926 cells and in A549 cells. The doubling time of EAhy926 cell and A549 cell proliferation was 25.32 h and 27.29 h, respectively (P > 0.1). Comparing the phase distribution of cell cycle of EAhy926 cells with that of A549 cells, the percentage of cells in G0/G1 phase, in S phase and in G2/M phase was (63.7% +/- 2.65%) VS (60.0% +/- 3.17%), (15.4% +/- 1.52%) VS (13.8% +/- 1.32%), and (20.9% +/- 3.40%) VS (26.3% +/- 3.17%), respectively (P > 0.05). For the ability of cell adhesion of EAhy926 cells and A549 cells, the value of OD in Eahy926 cells was significantly higher than that in A549 cells (0.3236 +/- 0.0514 VS 0.2434 +/- 0.0390, P < 0.004). We also found that the migration ability of Eahy926 cells was stronger than that of A549 cells (28.00 +/- 2.65 VS 18.00 +/- 1.00, P < 0.01), and that the invasion ability of Eahy926 cells was significantly weak than that of A549 cells (15.33 +/- 0.58 VS 26.67 +/- 2.52, P < 0.01). In the xenograft tumor model, expansive masses of classic tumor were found in the A549 cells

  3. Seven Murine Cell Lines with Properties of Macrophages,

    DTIC Science & Technology

    1981-02-01

    of this study; BALB-G-F, a fibroblast-like line derived from the same culture as BALB-G-M by cloning; L929 cells, a gift from Dr. Rolf Zinkernagel...less than 3% of cells ingested E under the same conditions. BW-J-T, NZW-D-T, BALB-G-T, BALB-G-F, L929 and TE-1 control cells were all nonphagocytic under...induced spreading. Exp. Cell Res. 79, 423, 1973. 30. Rabinovitch, M. and DeStefano, M. J. Use of the local anesthetic lidocaine for cell harvesting

  4. Phase transitions in tumor growth: II prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Llanos-Pérez, J. A.; Betancourt-Mar, A.; De Miguel, M. P.; Izquierdo-Kulich, E.; Royuela-García, M.; Tejera, E.; Nieto-Villar, J. M.

    2015-05-01

    We propose a mechanism for prostate cancer cell lines growth, LNCaP and PC3 based on a Gompertz dynamics. This growth exhibits a multifractal behavior and a "second order" phase transition. Finally, it was found that the cellular line PC3 exhibits a higher value of entropy production rate compared to LNCaP, which is indicative of the robustness of PC3, over to LNCaP and may be a quantitative index of metastatic potential tumors.

  5. Donor mesenchymal stem cells home to maternal wounds after transamniotic stem cell therapy (TRASCET) in a rodent model.

    PubMed

    Graham, Christopher D; Shieh, Hester F; Brazzo, Joseph A; Zurakowski, David; Fauza, Dario O

    2017-06-01

    Transamniotic stem cell therapy (TRASCET) with amniotic fluid-derived MSCs (afMSCs) has emerged experimentally as a practical treatment strategy for congenital anomalies. In this study, we sought to determine whether afMSCs migrate to the mother following TRASCET. Pregnant rat dams were divided into three groups. Two groups received volume-matched injections into all amniotic cavities of either a suspension of afMSCs labeled with a luciferase reporter gene or the luciferase protein alone. In a third group, a suspension of labeled cells was aliquoted onto the serosal surface of the uterus. Maternal samples from the laparotomy scar (fascia and skin separately), bone marrow, and peripheral blood were procured, along with placenta and umbilical cord. Specimens were screened for luminescence via microplate luminometry. Luminescence was detected in 60% (9/15) of the fascial scars from the group receiving intraamniotic injection of afMSCs, but in none of the other groups (P<0.001). There was a direct correlation between the presence of donor cells in the placenta and their presence in maternal fascia (Wald test=10.2; P=0.001). Amniotic mesenchymal stem cells migrate to maternal sites of injury after intraamniotic injection. Maternal homing of donor cells must be considered in the setting of transamniotic stem cell therapy. N/A (animal and laboratory study). Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Electrophysiological characterization of Nsc-34 cell line using Microelectrode Array.

    PubMed

    Sabitha, K R; Sanjay, D; Savita, B; Raju, T R; Laxmi, T R

    2016-11-15

    Neurons communicate with each other through intricate network to evolve higher brain functions. The electrical activity of the neurons plays a crucial role in shaping the connectivity. With motor neurons being vulnerable to neurodegenerative diseases, understanding the electrophysiological properties of motor neurons is the need of the hour, in order to comprehend the impairment of connectivity in these diseases. NSC-34 cell line serves as an excellent model to study the properties of motor neurons as they express Choline acetyltransferase (ChAT). Although NSC-34 cell lines have been used to study the effect of various toxicological, neurotrophic and neuroprotective agents, the electrical activity of these cells has not been elucidated. In the current study, we have characterized the electrophysiological properties of NSC-34 cell lines using Micro-Electrode Array (MEA) as a tool. Based on the spike waveform, firing frequency, auto- and cross-correlogram analysis, we demonstrate that NSC-34 cell culture has >2 distinct types of neuronal population: principal excitatory neurons, putative interneurons and unclassified neurons. The presence of interneurons in the NSC-34 culture was characterized by increased expression of GAD-67 markers. Thus, finding an understanding of the electrophysiological properties of different population of neurons in NSC-34 cell line, will have multiple applications in the treatment of neurological disorders.

  7. Cytokine profile of breast cell lines after different radiation doses.

    PubMed

    Bravatà, Valentina; Minafra, Luigi; Forte, Giusi Irma; Cammarata, Francesco Paolo; Russo, Giorgio; Di Maggio, Federica Maria; Augello, Giuseppa; Lio, Domenico; Gilardi, Maria Carla

    2017-09-01

    Ionizing radiation (IR) treatment activates inflammatory processes causing the release of a great amount of molecules able to affect the cell survival. The aim of this study was to analyze the cytokine signature of conditioned medium produced by non-tumorigenic mammary epithelial cell line MCF10A, as well as MCF7 and MDA-MB-231 breast cancer cell lines, after single high doses of IR in order to understand their role in high radiation response. We performed a cytokine profile of irradiated conditioned media of MCF10A, MCF7 and MDA-MB-231 cell lines treated with 9 or 23 Gy, by Luminex and ELISA analyses. Overall, our results show that both 9 Gy and 23 Gy of IR induce the release within the first 72 h of cytokines and growth factors potentially able to influence the tumor outcome, with a dose-independent and cell-line dependent signature. Moreover, our results show that the cell-senescence phenomenon does not correlate with the amount of 'senescence-associated secretory phenotype' (SASP) molecules released in media. Thus, additional mechanisms are probably involved in this process. These data open the possibility to evaluate cytokine profile as useful marker in modulating the personalized radiotherapy in breast cancer care.

  8. Caffeine augments Alprazolam induced cytotoxicity in human cell lines.

    PubMed

    Saha, Biswarup; Mukherjee, Ananda; Samanta, Saheli; Saha, Piyali; Ghosh, Anup Kumar; Santra, Chitta Ranjan; Karmakar, Parimal

    2009-09-01

    Combined effects of alprazolam (Alp), a member of benzodiazepine group of drugs and caffeine on human cell lines, HeLa and THP1 were investigated in this study. Alp mediated cytotoxicity was enhanced while caffeine was present. The cell death was confirmed by observing morphological changes, LDH assay and membrane anisotropic study. Also such combined effects induced elevated level of ROS and depletion of GSH. The mechanism of cell death induced by simultaneous treatment of Alp and caffeine was associated with the calcium-mediated activation of mu-calpain, release of lysosomal protease cathepsin B, activation of PARP and cleavage of caspase 3. Our results indicate that, Alp alone induces apoptosis in human cells but in the presence of caffeine it augments necrosis in a well-regulated pathway. Thus our observations strongly suggest that, alprazolam and caffeine together produce severe cytotoxicity in human cell lines.

  9. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    PubMed Central

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-01-01

    Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells. PMID:27916824

  10. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines.

    PubMed

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-11-29

    Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  11. Critical role of the α1-Na+,K+-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain

    PubMed Central

    Akimova, Olga A.; Tverskoi, Artem M.; Smolyaninova, Larisa V.; Mongin, Alexander A.; Lopina, Olga D.; La, Jennifer; Dulin, Nickolai O.; Orlov, Sergei N.

    2015-01-01

    SUMMARY In rodents, ubiquitous α1-Na+,K+-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~103-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na+,K+-ATPase isoforms, we compared the effects of ouabain on intracellular Na+ and K+ content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. Six-hour exposure of HASMC and HUVEC to 3 μM ouabain dramatically increased the intracellular [Na+]/[K+] ratio to the same extend as in RASMC and RAEC treated with 3,000 μM ouabain. In 24, 3 μM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3,000-5,000 μM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1R/R mouse, ouabain triggered death of MASMC from α1S/S mouse expressing human α1-Na+,K+-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na+,K+-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na+,K+-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery. PMID:26067145

  12. Rodent Research-1 Validation of Rodent Hardware

    NASA Technical Reports Server (NTRS)

    Globus, Ruth; Beegle, Janet

    2013-01-01

    To achieve novel science objectives, validation of a rodent habitat on ISS will enable - In-flight analyses during long duration spaceflight- Use of genetically altered animals- Application of modern analytical techniques (e.g. genomics, proteomics, and metabolomics)

  13. Generation and Characterization of JCV Permissive Hybrid Cell Lines

    PubMed Central

    Sariyer, Ilker K.; Safak, Mahmut; Gordon, Jennifer; Khalili, Kamel

    2009-01-01

    JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents. PMID:19442856

  14. Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines

    PubMed Central

    2012-01-01

    Background Many cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized. Methods Cell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20–80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines. Results The cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but

  15. Generation of cell lines for monoclonal antibody production.

    PubMed

    Alvin, Krista; Ye, Jianxin

    2014-01-01

    Monoclonal antibodies (mAbs) represent the largest group of therapeutic proteins with 30 products approved in the USA and hundreds of therapies currently undergoing clinical trials. The complex nature of mAbs makes their development as therapeutic agents constrained by numerous criteria such as quality, safety, regulation, and quantity. Identification of a clonal cell line expressing high levels of mAb with adequate quality attributes and generated in compliance with regulatory standards is a necessary step prior to a program moving to large-scale production for clinical material. This chapter outlines the stable transfection technology that generates clonal cell lines for commercial manufacturing processes.

  16. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    PubMed

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  17. Boldine: a potential new antiproliferative drug against glioma cell lines.

    PubMed

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent.

  18. Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3)-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv).

    PubMed

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Chow, Vincent T K; Chua, Kaw Bing

    2014-01-01

    Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

  19. Phenotypic and Genotypic Characteristics of Novel Mouse Cell Line (NIH/3T3)-Adapted Human Enterovirus 71 Strains (EV71:TLLm and EV71:TLLmv)

    PubMed Central

    Victorio, Carla Bianca Luena; Xu, Yishi; Ng, Qimei; Chow, Vincent T. K.; Chua, Kaw Bing

    2014-01-01

    Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus’s inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136–150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro. PMID:24671184

  20. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    SciTech Connect

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  1. Thyroid hormone transport in a human glioma cell line.

    PubMed

    Goncalves, E; Lakshmanan, M; Pontecorvi, A; Robbins, J

    1990-03-05

    The uptake of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) was studied in human glioma cells (Hs 683) and compared with that in several other neural cell lines. At 25 degrees C or 37 degrees C, total cell uptake rose rapidly and reached equilibrium within 60 min. The glioma cells had the highest uptake: 47.6 fmol of L-T3 and 43.4 fmol of L-T4 per 10(6) cells at 37 degrees C. These were inhibited 77% and 72%, respectively, by excess unlabeled hormone. Uptake in the nuclei reached equilibrium between 90 and 120 min and was also highest in glioma cells: 1.46 fmol of L-T3 and 0.49 fmol of L-T4 per 10(6) cells. When expressed as percent of total cell uptake, however, glioma cells had the lowest values (3.1% for L-T3 and 1.1% for L-T4). Also in contrast to other cell lines, glioma cells transported L-T4 almost as effectively as L-T3. D-T3 and D-T4 total cell uptake was 86% and 96% lower than that of the respective L-isomers, and the nuclear uptake as a fraction of the cell uptake was similar. Kinetic analysis of the initial rate of cell uptake gave Vmax values for D-T3 and D-T4 that were 97% and 98% lower than for the L-isomers. Antimycin and monodansylcadaverine decreased the Vmax as well as the equilibrium cell and nuclear uptake of the L-isomers. The apparent nuclear affinity constant for L-T4 in intact cells was inhibited 90% in the presence of antimycin, whereas no effect was observed in isolated nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Apparent reduction of ADAM10 in scrapie-infected cultured cells and in the brains of scrapie-infected rodents.

    PubMed

    Chen, Cao; Lv, Yan; Zhang, Bao-Yun; Zhang, Jin; Shi, Qi; Wang, Jing; Tian, Chan; Gao, Chen; Xiao, Kang; Ren, Ke; Zhou, Wei; Dong, Xiao-Ping

    2014-12-01

    It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP(C)) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP(C). Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP(C). Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.

  3. Rodent host cell/Lassa virus interactions: evolution and expression of α-Dystroglycan, LARGE-1 and LARGE-2 genes, with special emphasis on the Mastomys genus.

    PubMed

    Tayeh, Ashraf; Tatard, Caroline; Kako-Ouraga, Sandrine; Duplantier, Jean-Marc; Dobigny, Gauthier

    2010-12-01

    Arenaviruses are usually rodent-borne viruses that constitute a major threat for human health. Among them, Lassa Fever Virus (LFV) occurs in Western Africa where it infects hundreds of thousands of people annually. According to the most recent surveys, LFV is hosted by one of the multimammate rats, Mastomys natalensis, but has never been detected in its sibling and sometimes sympatric species Mastomys erythroleucus. This pattern suggests that intrinsic, i.e. genetic properties underlie such a drastic epidemiological difference (M. natalensis as a reservoir vs. M. erythroleucus as a non-reservoir species). Here we investigate genomic differences between these two closely related rodent species by focusing on three genes that have recently been described as pivotal for LFV/human cell interactions: Dystroglycan (the LFV cellular receptor), LARGE-1 and LARGE-2 (two enzymes that are essential to Dystroglycan functioning). For all three genes, sequence analyses showed that amino-acid chains undergo extremely strong purifying selective pressures, and indicated that no nucleotide (therefore no tertiary structure) change can be advocated to explain species-specific differences in LFV-cellular mediation. Nevertheless, preliminary studies of kidney-specific expression profiles suggested that important species-specific differences exist between Mastomys species. Taking into account current knowledge about LFV-human cell interactions, our results may point towards a possible role for LARGE-1 and LARGE-2 enzymes at the intracellular replication level of the virus, rather than at the LFV-host cell receptor binding step.

  4. Increased EGF receptors on human squamous carcinoma cell lines.

    PubMed Central

    Cowley, G. P.; Smith, J. A.; Gusterson, B. A.

    1986-01-01

    Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours. PMID:2420349

  5. Mouse DRG Cell Line with Properties of Nociceptors.

    PubMed

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C; Grundy, David; Nassar, Mohammed A

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

  6. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

    PubMed Central

    Krampe, Britta

    2010-01-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture. PMID:20502964

  7. Transthyretin expression in medulloblastomas and medulloblastoma cell lines.

    PubMed

    Albrecht, S; Bayer, T A; Kraus, J A; Pietsch, T

    1995-10-01

    Transthyretin is a protein crucial to the transport of lipophilic molecules such as thyroid hormones and retinoids. In the central nervous system, large amounts of transthyretin are synthesized by the choroid plexus and are secreted into the cerebrospinal fluid. The choroid plexus is the only site of transthyretin synthesis in the brain. Transthyretin is expressed by most benign and malignant choroid plexus tumours while gliomas and meningiomas do not express transthyretin. Other major sites of transthyretin synthesis are the retinal pigment epithelium and hepatocytes. Medulloblastoma is the prototypical primitive neuroectodermal tumour of the cerebellum and can show multiple lines of differentiation, including the expression of retinal markers. In this study, we examined transthyretin expression both at the RNA and protein level in four medulloblastomas and six medulloblastoma cell lines using Northern and Western blot analysis, reverse transcription polymerase chain reaction (PCR), RNA in situ hybridization, and immunohistochemistry. All four medulloblastomas and five of the six medulloblastoma cell lines expressed transthyretin-mRNA as demonstrated by reverse PCR and in situ hybridization while three medulloblastomas and one cell line were positive on Northern blot. The medulloblastoma with the most abundant RNA expression was transthyretin-immunoreactive on cryosections and the medulloblastoma cell line that was positive on Northern blot also expressed transthyretin at levels detectable by Western blot. No transthyretin-immunoreactivity was seen in 16 additional medulloblastomas studied on paraffin sections. These findings indicate that low-level expression of transthyretin-mRNA is common in medulloblastomas and medulloblastoma cell lines. Expression of transthyretin protein occurs rarely but can reach significant levels. Transthyretin expression in medulloblastoma is consistent with retinal pigment epithelium differentiation in medulloblastomas and reflects

  8. Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

    PubMed Central

    Coram, Marc A.; Reddy, Anupama; Deng, Glenn; Telli, Melinda L.; Advani, Ranjana H.; Carlson, Robert W.; Mollick, Joseph A.; Sheth, Shruti; Kurian, Allison W.; Ford, James M.; Stockdale, Frank E.; Quake, Stephen R.; Pease, R. Fabian; Mindrinos, Michael N.; Bhanot, Gyan; Dairkee, Shanaz H.; Davis, Ronald W.; Jeffrey, Stefanie S.

    2012-01-01

    Background To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs

  9. Engineering Retina from Human Retinal Progenitors (Cell Lines)

    PubMed Central

    Cao, Yang

    2009-01-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell–cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr2e3, expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription–polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell

  10. The silver lining of induced pluripotent stem cell variation

    PubMed Central

    Jain, Tanya; Sevilla, Ana

    2016-01-01

    Induced pluripotent stem cells (iPSCs) are being generated using various reprogramming methods and from different cell sources. Hence, a lot of effort has been devoted to evaluating the differences among iPSC lines, in particular with respect to their differentiation capacity. While line-to-line variability should mainly reflect the genetic diversity within the human population, here we review some studies that have brought attention to additional variation caused by genomic and epigenomic alterations. We discuss strategies to evaluate aberrant changes and to minimize technical and culture-induced noise, in order to generate safe cells for clinical applications. We focus on the findings from a recent study, which compared the differentiation capacity of several iPSC lines committed to the hematopoietic lineage and correlated the differential maturation capacity with aberrant DNA methylations. Although iPSC variation represents a challenge for the field, we embrace the authors’ perspective that iPSC variations should be used to our advantage for predicting and selecting the best performing iPSC lines, depending on the desired application. PMID:28066788

  11. Maslinic Acid Inhibits Proliferation of Renal Cell Carcinoma Cell Lines and Suppresses Angiogenesis of Endothelial Cells

    PubMed Central

    Thakor, Parth; Song, Wenzhe; Subramanian, Ramalingam B.; Thakkar, Vasudev R.; Vesey, David A.

    2017-01-01

    Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC) remains a treatment-resistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1), endothelial cells (human umbilical vein endothelial cell line [HUVEC]), and primary cultures of kidney proximal tubular epithelial cells (PTEC) were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy. PMID:28405545

  12. Volatile metabolomic signature of human breast cancer cell lines.

    PubMed

    Silva, Catarina L; Perestrelo, Rosa; Silva, Pedro; Tomás, Helena; Câmara, José S

    2017-03-03

    Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC-MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways.

  13. Volatile metabolomic signature of human breast cancer cell lines

    PubMed Central

    Silva, Catarina L.; Perestrelo, Rosa; Silva, Pedro; Tomás, Helena; Câmara, José S.

    2017-01-01

    Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC–MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways. PMID:28256598

  14. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  15. Antiproliferative Properties of Clausine-B against Cancer Cell Lines

    PubMed Central

    Wan Mohd Zain, Wan Nor I’zzah; Rahmat, Asmah; Othman, Fauziah; Yap, Taufiq Yun Hin

    2009-01-01

    Background: Clausine B, a carbazole alkaloid isolated from the stem bark of Clausena excavata, was investigated for its antiproliferative activities against five human cancer cell lines: HepG2 (hepatic cancer), MCF-7 (hormone-dependent breast cancer), MDA-MB-231 (non-hormone-dependent breast cancer), HeLa (cervical cancer), and CAOV3 (ovarian cancer). Methods: Chang liver (normal cells) was used as a control. The effect of clausine-B was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results: Clausine-B was found to be active (IC50<30 μg/mL) against four of the cancer cell lines tested. The IC50 values for these four lines were: 21.50 μg/mL (MDA-MB-231), 22.90 g/ml (HeLa), 27.00 μg/mL (CAOV3) and 28.94 μg/mL (HepG2). Clausine-B inhibited the MCF-7 cancer cell line at 52.90 μg/mL, and no IC50 value was obtained against Chang liver. Conclusion: It is possible that the phenolic group in clausine-B responsible for the antiproliferative activities found in this study. PMID:22589662

  16. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  17. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  18. Use of Cell Lines in the Investigation of Pharmacogenetic Loci

    PubMed Central

    Zhang, Wei; Dolan, M. Eileen

    2009-01-01

    Drug response and toxicity, complex traits that are often highly varied among individuals, likely involve multiple genetic and non-genetic factors. Pharmacogenomic research aims to individualize therapy in an effort to maximize efficacy and minimize toxicity for each patient. Cell lines can be used as a model system for cellular pharmacologic effects, which include, but are not limited to, drug-induced cytotoxicity or apoptosis, biochemical effects and enzymatic reactions. Because severe toxicities may be associated with drugs such as chemotherapeutics, cell lines derived from healthy individuals or patients provide a convenient model to study how human genetic variation alters response to these drugs that would be unsafe or unethical to administer to human volunteers. In addition to the traditional candidate gene approaches that focus on well-understood candidate genes and pathways, the availability of extensive genotypic and phenotypic data on some cell line models has begun to allow genome-wide association (GWA) studies to simultaneously test the entire human genome for associations with drug response and toxicity. Though with some important limitations, the use of these cell lines in pharmacogenomic discovery demonstrates the promise of constructing a more comprehensive model that may ultimately integrate both genetic and non-genetic factors to predict individual response and toxicity to anticancer drugs. PMID:19925429

  19. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... identification as part of this project will undergo STR profiling, a DNA profiling method that examines/screens for STRs (DNA elements 2-6 bps long repeated in tandem) in the human chromosomes, that has been shown... are expected between cell line DNA samples originating from unrelated individuals. Each unique...

  20. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  1. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    PubMed

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. © The Author 2016. Published by Oxford University Press on behalf of Poultry Science Association.

  2. Curbing rampant cross-contamination and misidentification of cell lines.

    PubMed

    Nardone, Roland M

    2008-09-01

    A son's challenge started an emeritus professor of biology on a three-year odyssey to get biological researchers to correct a decades-long problem with cross-contaminated and misidentified cell lines. These errors may account for more than 15% of mammalian cultures, wasting resources and undermining the integrity of research.

  3. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  4. Radiation-induced delayed cell death in a hypomorphic Artemis cell line.

    PubMed

    Evans, Paul M; Woodbine, Lisa; Riballo, Enriquetta; Gennery, Andrew R; Hubank, Michael; Jeggo, Penny A

    2006-04-15

    Null mutations in Artemis confer a condition described as RS-SCID, in which patients display radiosensitivity combined with severe combined immunodeficiency. Here, we characterize the defect in Artemis in a patient who displayed progressive combined immunodeficiency (CID) and elevated lymphocyte apoptosis. The patient is a compound heterozygote with novel mutations in both alleles, resulting in Artemis proteins with either L70 deletion or G126D substitution. Both mutational changes impact upon Artemis function and a fibroblast cell line derived from the patient (F96-224) has greatly reduced Artemis protein. In contrast to Artemis null cell lines, which fail to repair a subset of DNA double strand breaks (DSBs) induced by ionizing radiation, F96-224 cells show slow but residual DSB rejoining. Despite showing intermediate cellular and clinical features, F96-224 cells are as radiosensitive as Artemis null cell lines. We developed a FACS-based assay to examine cell division and cellular characteristics for 10 days following exposure to ionizing radiation (2 and 4 Gy). This analysis demonstrated that F96-224 cells show delayed cell death when compared with rapid growth arrest of an Artemis null cell line, and the emergence of a cycling population shown by a control line. F96-224 cells also display elevated chromosome aberrations when compared with control cells. F96-224 therefore represents a novel phenotype for a hypomorphic cell line. We suggest that delayed cell death contributes to the progressive CID phenotype of the Artemis patient.

  5. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as described...

  6. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations

    PubMed Central

    Meeth, Katrina; Wang, Jake; Micevic, Goran; Damsky, William; Bosenberg, Marcus W.

    2017-01-01

    Summary The remarkable success of immune therapies emphasizes the need for immune competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here we describe a comprehensive system of mouse melanoma cell lines that are syngeneic to C57Bl/6J, have well-defined human-relevant driver mutations, and are genomically stable. These will be a useful tool for the study of tumor immunology and genotype-specific cancer biology. PMID:27287723

  7. L1 expression and regulation in humans and rodents

    PubMed Central

    Rosser, James M.; An, Wenfeng

    2015-01-01

    Long interspersed elements type 1 (LINE-1s, or L1s) have impacted mammalian genomes at multiple levels. L1 transcription is mainly controlled by its 5’ untranslated region (5’UTR), which differs significantly among active human and rodent L1 families. In this review, L1 expression and its regulation are examined in the context of human and rodent development. First, endogenous L1 expression patterns in three different species—human, rat, and mouse—are compared and contrasted. A detailed account of relevant experimental evidence is presented according to the source material, such as cell lines, tumors, and normal somatic and germline tissues from different developmental stages. Second, factors involved in the regulation of L1 expression at both transcriptional and posttranscriptional levels are discussed. These include transcription factors, DNA methylation, PIWI-interacting RNAs (piRNAs), RNA interference (RNAi), and posttranscriptional host factors. Similarities and differences between human and rodent L1s are highlighted. Third, recent findings from transgenic mouse models of L1 are summarized and contrasted with those from endogenous L1 studies. Finally, the challenges and opportunities for L1 mouse models are discussed. PMID:22202032

  8. Establishment a CHO Cell Line Expressing Human CD52 Molecule

    PubMed Central

    Kadijeh, Tati; Mahsa, Yazdanpanah-Samani; Amin, Ramezani; Elham, Mahmoudi Maymand; Abbas, Ghaderi

    2016-01-01

    Background: CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details Methods: Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. Results: Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. Conclusion: In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids PMID:28070536

  9. Immortality of cell lines: challenges and advantages of establishment.

    PubMed

    Maqsood, Muhammad Irfan; Matin, Maryam M; Bahrami, Ahmad Reza; Ghasroldasht, Mohammad M

    2013-10-01

    Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.There are also some other factors and mechanisms involved in immortalisation, which need to be discovered. Immortalisation of cells derived from different sources and establishment of immortal cell lines has proven useful in understanding the molecular pathways governing cell developmental cascades in eukaryotic, especially human, cells. After the breakthrough of achieving the immortal cells and understanding their critical importance in the field of molecular biology, intense efforts have been dedicated to establish cell lines useful for elucidating the functions of telomerase, developmental lineage of progenitors, self-renewal potency, cellular transformation, differentiation patterns and some bioprocesses, like odontogenesis. Meanwhile, discovering the exact mechanisms of immortality, a major challenge for science yet, is believed to open new gateways toward understanding and treatment of cancer in the long term. This review summarises the methods involved in establishing immortality, its advantages and the challenges still being faced in this field.

  10. The effect of sclareol on growth and cell cycle progression of human leukemic cell lines.

    PubMed

    Dimas, K; Kokkinopoulos, D; Demetzos, C; Vaos, B; Marselos, M; Malamas, M; Tzavaras, T

    1999-03-01

    Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.

  11. Biological characteristics of side population cells in a self-established human ovarian cancer cell line

    PubMed Central

    WEI, ZHENTONG; LV, SHUANG; WANG, YISHU; SUN, MEIYU; CHI, GUANGFAN; GUO, JUN; SONG, PEIYE; FU, XIAOYU; ZHANG, SONGLING; LI, YULIN

    2016-01-01

    The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×103 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self

  12. Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines

    PubMed Central

    2014-01-01

    Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 μg/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p < 0.01) in cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the

  13. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing.

    PubMed

    Quentmeier, Hilmar; Drexler, Hans G; Hauer, Vivien; MacLeod, Roderick A F; Pommerenke, Claudia; Uphoff, Cord C; Zaborski, Margarete; Berglund, Mattias; Enblad, Gunilla; Amini, Rose-Marie

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.

  14. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing

    PubMed Central

    Quentmeier, Hilmar; Drexler, Hans G.; Hauer, Vivien; MacLeod, Roderick A. F.; Pommerenke, Claudia; Uphoff, Cord C.; Zaborski, Margarete; Berglund, Mattias

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members. PMID:27907212

  15. Leukemia L1210 cell lines resistant to ribonucleotide reductase inhibitors.

    PubMed

    Cory, J G; Carter, G L

    1988-02-15

    Leukemia L1210 cell lines, ED1 and ED2, were generated which were resistant to the cytotoxic effects of deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine and deoxyadenosine/erythro-9-(2-hydroxyl-3-nonyl)adenine plus 2,3-dihydro-1H-pyrazole[2,3a]imidazole/Desferal, respectively. The ED1 and ED2 were characterized to show that these cell lines had increased levels of ribonucleotide reductase as measured by CDP reduction. The reductase activity in crude cell-free extracts from the ED1 and ED2 cells was not inhibited by dATP. For CDP reductase, the activation by adenylylimido diphosphate and inhibition by dGTP and dTTP in these extracts from the ED1 and ED2 cells were the same as for the wild-type L1210 cells. The ED1 and ED2 cells were highly cross-resistant, as measured by growth inhibition, to deoxyguanosine/8-aminoguanosine, 2-fluorodeoxyadenosine, and 2-fluoroadenine arabinoside. While the ED2 cells showed resistance to 2,3-dihydro-1H-pyrazole-[2,3a]-imidazole/Desferal (6-fold), the ED1 and ED2 cell lines showed less resistance to hydroxyurea, 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone, and the dialdehyde of inosine. These data indicate that the mechanisms of resistance to the ribonucleotide reductase inhibitors are related to the increased level of ribonucleotide reductase activity and to the decreased sensitivity of the effector-binding subunit to dATP.

  16. Zebrafish kidney stromal cell lines support multilineage hematopoiesis

    PubMed Central

    Stachura, David L.; Reyes, Jason R.; Bartunek, Petr; Paw, Barry H.; Zon, Leonard I.

    2009-01-01

    Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish. PMID:19433857

  17. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    SciTech Connect

    Heaton, D.; Mustafi, R.; Schwartz, J.L. |

    1992-06-01

    The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

  18. Species peculiarities in damage to regulatory systems of murine rodents` liver cells in conditions of slight radioactive contamination

    SciTech Connect

    Kudyasheva, A.G.; Shishkina, L.N.; Zagorskaya, N.G.

    1995-07-01

    Results are given from comparative analysis of the antioxidation activity (AOA) of lipids, composition of phospholipids, and activity of Krebs`-cycle and glycolysis enzymes in the liver of three species of murine rodents caught in the slightly contaminated zone of the accident at the Chernobyl nuclear power plant. Disruptions were found in individual links of the regulation of processes of peroxidation of lipids (POL), as well as depression and discoordination of dehydrogenation process. The sharpest shifts in biochemical and biophysical indices were noted in the more radiosensitive root vole.

  19. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    PubMed Central

    2011-01-01

    Background Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs. PMID:21669008

  20. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines.

    PubMed

    Kowarz, Eric; Löscher, Denise; Marschalek, Rolf

    2015-04-01

    Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Fucose-targeted glycoengineering of pharmaceutical cell lines.

    PubMed

    Ogorek, Christiane; Jordan, Ingo; Sandig, Volker; von Horsten, Hans Henning

    2012-01-01

    Glycosylation is known to have an impact on pharmacokinetics and pharmacodynamics of therapeutic proteins. While the production of pharmaceutically desirable glycosylation forms of a therapeutic protein can in certain cases be influenced by the upstream process parameters, certain specialized glycan structures can only be produced in large quantities from cell lines that have been genetically engineered.One particular case where a specialized glycostructure has a major impact on pharmacodynamic mode of action is the enhanced ADCC-effector function of afucosylated IgG1-type monoclonal antibodies. Here we describe the methodological details of a powerful yet simple glycoengineering approach targeted at the fucosylation machinery within eukaryotic cells. As an example we demonstrate the modification of the permanent avian cell line AGE1.CR.pIX which is characterized by a unique glycosylation machinery.

  2. Androglobin knockdown inhibits growth of glioma cell lines

    PubMed Central

    Huang, Bo; Lu, Yi-Sheng; Li, Xia; Zhu, Zhi-Chuan; Li, Kui; Liu, Ji-Wei; Zheng, Jing; Hu, Ze-Lan

    2014-01-01

    Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro. PMID:24966926

  3. Plasmids and packaging cell lines for use in phage display

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  4. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  5. [Sorting of side population cells from multiple myeloma cell lines and analysis of their biological characteristics].

    PubMed

    Zhang, Xiao-Li; Zhang, Li-Na; Huang, Hong-Ming; Ding, Run-Sheng; Shi, Wei; Xu, Rui-Rong; Yu, Xiao-Tang; Jiang, Sheng-Hua

    2014-06-01

    This study was aimed to sort the side population (SP) cells from human multiple myeloma cell lines, then detect the biological characteristics of those SP cells. After Hoechst33342 staining, intracellular Hoechst33342 fluorescence staining differences of myeloma cell lines observed by the fluorescence microscopy. The fluorescence-activated cell sorting (FACS) technology was used to isolate SP cells and main population (MP) cells; proliferative capacity in vitro was determined by cell growth curve; the cell colony forming ability was compared by colony forming test. The CD138 expression was detected by flow cytometry. The expression of ABCG2 mRNA was detected by reverse transcription PCR; CCK-8 assay and colony forming test were used to evaluate the effect of bortezomib on the cell proliferation, vitality and colony forming ability of the two populations. The results showed that the myeloma cell lines had a small proportion of SP cells, especially, RPMI 8226 cells accounted for the highest proportion of SP cells (7.10 ± 2.69)%, which have also been confirmed under the fluorescence microscope; the proliferative activity and cell colony forming ability of SP cells were significantly higher than those of MP cells (P < 0.05). The expression levels of CD138 in SP and MP cells were not significantly different (P > 0.05). RT-PCR results showed that SP cells expressed the drug-resistance gene ABCG2, but MP cells hardly express these genes. The inhibition rate of bortezomib on SP cells was significantly lower than that on MP cells (P < 0.05), however, the difference was not significant (P > 0.05) at bortezomib 40 nmol/L. Bortezomib could reduce colony formation in the both two cell populations, but more severe reduction appeared in the MP cells. It is concluded that the myeloma cell line contain a small amount of SP cells with the cancer stem cell characteristics.

  6. Establishment and characterization of human non-small cell lung cancer cell lines.

    PubMed

    Li, Jiangchao; Yang, Hong; Chen, Leilei; Li, Yan; Zhu, Yinghui; Dai, Yongdong; Chen, Kai; Ai, Jiaoyu; Zeng, Tingting; Mao, Xueying; Liu, Lulu; Li, Xiaodong; Guan, Xin-Yuan

    2012-01-01

    Non-small cell lung cancer (NSCLC), a highly malignant tumor, is common in China and is associated with a very poor 5-year survival rate. To better understand the cancer biology of this disease, we report here the establishment of three new NSCLC cell lines, SCC210011, SCC211441 and ACC212102, from the tumor tissue of three NSCLC patients. By histological analysis, we found that all three cell lines displayed the typical features of endothelial cancer cells. The population doubling times of SCC210011, SCC211441 and ACC212102 cells were 42, 38 and 25 h, respectively. Our cytogenetic studies indicated that these cell lines exhibit structural and numerical chromosomal abnormalities. Furthermore, the tumorigenicity in nude mice was confirmed, and H&E staining results revealed that they resembled the primary tissue. These newly established cell lines may serve as useful models for studying the molecular pathogenesis of NSCLC.

  7. Heterogeneity of a human T-lymphoblastoid cell line

    SciTech Connect

    Snow, K.; Judd, W.

    1987-08-01

    A human T-lymphoblastoid cell line (Jurkat) was cloned, and four resulting sublines were characterized in a variety of ways with the objective of gaining information on heterogeneity in cell lines. Within a few weeks of cloning, distinct cellular morphologies and growth patterns became apparent in the four sublines. Growth rate measurements made over 3 months did not show any significant differences between the sublines. Surface protein profiles obtained by radioimmunoprecipitation using antisera in conjunction with extracts from (/sup 35/S)Met and /sup 125/I-labeled cells revealed differences between the sublines. Analysis of total cell DNA showed that one of the sublines possessed only half the chromosome complement of the other sublines and the parental line. Karyotyping confirmed this result and, in addition, demonstrated that chromosome numbers fluctuated around a mean value for each subline. Karyotypic variability became apparent within 2 months of cloning and tended to increase with time in culture. G-banding analysis showed that the analyzed cell populations contained distinctive cytogenetic aberrations. Properties of the cloned sublines were monitored over a 9-month period. One of the sublines that had shown heterogeneous morphology even after 6 weeks maintained the heterogeneity throughout this time. Another subline underwent a marked change in morphology (round to irregular) and growth habit (single cells to large clumps) with increasing time in culture. Interestingly, several alterations to surface proteins accompanied these growth changes. A third subline had relatively stable morphology and chromosome number throughout the 9-month period. The modal chromosome number was hypotetraploid for three sublines and the parent line, but was diploid for another subline.

  8. Comparative Proteomic Profiling of Pancreatic Ductal Adenocarcinoma Cell Lines

    PubMed Central

    Kim, Yikwon; Han, Dohyun; Min, Hophil; Jin, Jonghwa; Yi, Eugene C.; Kim, Youngsoo

    2014-01-01

    Pancreatic cancer is one of the most fatal cancers and is associated with limited diagnostic and therapeutic modalities. Currently, gemcitabine is the only effective drug and represents the preferred first-line treatment for chemotherapy. However, a high level of intrinsic or acquired resistance of pancreatic cancer to gemcitabine can contribute to the failure of gemcitabine treatment. To investigate the underlying molecular mechanisms for gemcitabine resistance in pancreatic cancer, we performed label-free quantification of protein expression in intrinsic gemcitabine-resistant and - sensitive human pancreatic adenocarcinoma cell lines using our improved proteomic strategy, combined with filter-aided sample preparation, single-shot liquid chromatography-mass spectrometry, enhanced spectral counting, and a statistical method based on a power law global error model. We identified 1931 proteins and quantified 787 differentially expressed proteins in the BxPC3, PANC-1, and HPDE cell lines. Bioinformatics analysis identified 15 epithelial to mesenchymal transition (EMT) markers and 13 EMT-related proteins that were closely associated with drug resistance were differentially expressed. Interestingly, 8 of these proteins were involved in glutathione and cysteine/methionine metabolism. These results suggest that proteins related to the EMT and glutathione metabolism play important roles in the development of intrinsic gemcitabine resistance by pancreatic cancer cell lines. PMID:25518923

  9. Connexin 43 enhances paclitaxel cytotoxicity in colorectal cancer cell lines

    PubMed Central

    Wang, Siqi; Zhang, Shiwu; Zhao, Zhenying; Zhang, Chunze; Yang, Xiaoyun; Wang, Yijia

    2017-01-01

    Colorectal cancer has a relatively low sensitivity to paclitaxel. The purpose of this study was to investigate the role of connexin 43 (Cx43), which is a structural component of gap junctional communication (GJC), in paclitaxel cytotoxicity in colorectal cancer cells. Three colorectal cancer cell lines (HCT106, HCT116 and LoVo) were transfected with Cx43 and used to examine paclitaxel cytotoxicity. A western blot assay was used to confirm Cx43 expression in transfected cell lines as well as the expression of several proteins that are associated with paclitaxel cytotoxicity. A parachute dye-coupling assay was used to measure GJC function. An MTT assay was used to analyze the viability of paclitaxel-treated cells. Cx43 expression level and GJC function were significantly upregulated by the transfection (P<0.05). The viability of transfected cells was significantly inhibited compared with that of untransfected cells when treated with paclitaxel (20 or 80 nM) at high culture density but not at low culture density (P<0.05). Cx43 transfection significantly increased the mitotic arrest, tubulin polymerization and apoptosis effects of paclitaxel (P<0.05). It was also found that paclitaxel had an inhibitory effect on GJC function after 12 h of treatment in LoVo cells (P<0.05). These results indicate that Cx43 may serve as a target of paclitaxel chemotherapy for colorectal cancer. PMID:28810580

  10. Spontaneous malignant transformation in two epithelial cell lines of rat liver cells.

    PubMed

    Masuji, H; Sato, J

    1976-10-01

    The cellular morphology, chromosomal structure, and tumorigenicity of two lines (B and J-13) of rat epithelial cells were examined serially during in vitro cultivation. The cells for such cultures were derived from the hepatic tissues of two 7-day-old male rats of the Donryu strain. The cultured cells were first inoculated into newborn syngenetic rats on the 641st day in vitro (80th subcultures) for line B, and on the 446th day (58 subcultures) for line J-13. The inoculated cells produced tumors with hemorrhagic ascites in rats after long latent periods, viz, 215-599 days in line B and 170-369 days in line J-13. All the tumors were undifferentiated hepatocarcinomas. The pleomorphism in shape and size of the cultured cells gradually became obvious with time of cultivation and was more pronounced in recultured tumor cells. Chromosomes of the culured cells were a normal diploid pattern until about the 200th day in vitro, but thereafter the modal chromsome number shifted to hypodiploid or hypotriploid via hypodiploid stages. The chromosome constitution of recultured tumor cells resembled that of inoculated cells in number distribution, but had changed to a more complicated karyotype. In experiments with line B, the same marker chromosome was detected in all tumor cells analyzed as had been present in inoculated cells.

  11. Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines.

    PubMed

    Lopez-Campistrous, Ana; Song, Xiaohua; Schrier, Adam J; Wender, Paul A; Dower, Nancy A; Stone, James C

    2012-08-01

    The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim(-) mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim(+) cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim(-)) and Daudi (Bim(+)), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases. Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  12. Cytotoxicity evaluation of silica nanoparticles using fish cell lines.

    PubMed

    Vo, Nguyen T K; Bufalino, Mary R; Hartlen, Kurtis D; Kitaev, Vladimir; Lee, Lucy E J

    2014-01-01

    Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO2 NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO2 NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100 μg/mL were needed to achieve 24 h EC50 values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO2 NPs (16 nm) were relatively more toxic than larger sized ones (24 and 44 nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC50 values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays.

  13. Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

    PubMed

    Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

    2016-12-12

    The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 9999: 1-15, 2016. © 2016 Wiley Periodicals, Inc.

  14. Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines

    PubMed Central

    Ban, Sadayuki; Michikawa, Yuichi; Ishikawa, Ken-ichi; Sagara, Masashi; Watanabe, Koji; Shimada, Yutaka; Inazawa, Johji; Imai, Takashi

    2005-01-01

    The purpose of this study was to determine the radiosensitivities of 31 human oesophageal squamous cell carcinoma cell lines with a colony-formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. Ataxia-telangiectasia-mutated (ATM) gene in these cells had five missense mutations, and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non-hereditary diseased patients must be a good target for radiotherapy. PMID:16045545

  15. Bioenergetic Analysis of Ovarian Cancer Cell Lines: Profiling of Histological Subtypes and Identification of a Mitochondria-Defective Cell Line

    PubMed Central

    Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L. P. Madhubhani P.; Uusitalo, Larissa M.; Hempel, Nadine

    2014-01-01

    Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that

  16. Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line.

    PubMed

    Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L P Madhubhani P; Uusitalo, Larissa M; Hempel, Nadine

    2014-01-01

    Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that

  17. Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo.

    PubMed

    Moon, Jisook; Schwarz, Sigrid C; Lee, Hyun-Seob; Kang, Jun Mo; Lee, Young-Eun; Kim, Bona; Sung, Mi-Young; Höglinger, Günter; Wegner, Florian; Kim, Jin Su; Chung, Hyung-Min; Chang, Sung Woon; Cha, Kwang Yul; Kim, Kwang-Soo; Schwarz, Johannes

    2017-02-01

    We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening. Stem Cells Translational Medicine 2017;6:576-588.

  18. A protein crosslinking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments

    PubMed Central

    Boudreau, Amy C.; Milovanovic, Mike; Conrad, Kelly L.; Nelson, Christopher; Ferrario, Carrie R.; Wolf, Marina E.

    2012-01-01

    Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then cell surface-expressed receptors are covalently crosslinked to nearby proteins using the membrane-impermeable, bifunctional crosslinker bis(sulfosuccinimidyl)suberate (BS3). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after crosslinking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants. PMID:22470150

  19. Simultaneous quantification of the glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) receptor agonists in rodent plasma by on-line solid phase extraction and LC-MS/MS.

    PubMed

    Wang, Yan; Roth, Jonathan D; Taylor, Steven W

    2014-04-15

    Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) and the cholecystokinin-1 receptor (CCK1-R) have therapeutic potential because of their marked anorexigenic and weight lowering effects. Furthermore, recent studies in rodents have shown that co-administration of these agents may prove more effective than treatment either of the peptide classes alone. To correlate the pharmacodynamic effects to the pharmacokinetics of these peptide drugs in vivo, a sensitive and robust bioanalytical method is essential. Furthermore, the simultaneous determination of both analytes in plasma samples by a single method offers obvious advantages. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is well suited to this goal through its ability to simultaneously monitor multiple analytes through selected reaction monitoring (SRM). However, it is a challenge to find appropriate conditions that allow two peptides with widely disparate physiochemical properties to be simultaneously analyzed while maintaining the necessary sensitivity for their accurate plasma concentrations. Herein, we report an on-line solid phase extraction (SPE) LC-MS/MS method for simultaneous quantification of the CCK1-R agonist AC170222 and the GLP-1R agonist AC3174 in rodent plasma. The assay has a linear range from 0.0975 to 100ng/mL, with lower limits of quantification of 0.0975ng/mL and 0.195ng/mL for AC3174 and AC170222, respectively. The intra- and inter-day precisions were below 15%. The developed LC-MS/MS method was used to simultaneously quantify AC3174 and AC170222, the results showed that the terminal plasma concentrations of AC3174 or AC170222 were comparable between groups of animals that were administered with the peptides alone (247±15pg/mL of AC3174 and 1306±48pg/mL of AC170222), or in combination (222±32pg/mL and 1136±47pg/mL of AC3174 and AC170222, respectively). These data provide information on the drug exposure to aid in assessing the combination effects of AC3174 and AC

  20. Neurofilament protein aggregation in a cell line model system.

    PubMed

    Hull, Elizabeth; Spoja, Christoffer; Cordova, Matt; Cohlberg, Jeffrey A

    2008-02-01

    Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.

  1. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines

    PubMed Central

    Contino, Gianmarco; Eldridge, Matthew D.; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G.; Edwards, Paul A.W.; Fitzgerald, Rebecca C.

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. PMID:27594985

  2. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    PubMed

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

  3. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    USDA-ARS?s Scientific Manuscript database

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  4. A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

    PubMed

    Auffray, I; Dubart, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-05-01

    In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or

  5. Rosiglitazone inhibits cell proliferation by inducing G1 cell cycle arrest and apoptosis in ADPKD cyst-lining epithelia cells.

    PubMed

    Liu, Yawei; Dai, Bing; Fu, Lili; Jia, Jieshuang; Mei, Changlin

    2010-06-01

    Abnormal proliferation is an important pathological feature of autosomal dominant polycystic kidney disease (ADPKD). Many drugs inhibiting cell proliferation have been proved to be effective in slowing the disease progression in ADPKD. Recent evidence has suggested that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have anti-neoplasm effects through inhibiting cell growth and inducing cell apoptosis in various cancer cells. In the present study, we examined the expression of PPARgamma in human ADPKD kidney tissues and cyst-lining epithelial cell line, and found that the expression of PPARgamma was greater in ADPKD kidney tissues and cyst-lining epithelial cell line than in normal kidney tissues and human kidney cortex (HKC) cell line. Rosiglitazone inhibited significantly proliferation of cyst-lining epithelial cells in a concentration- and time-dependent manner. These effects were diminished by GW9662, a specific PPARgamma antagonist. Cell cycle analysis showed a G0/G1 arrest in human ADPKD cyst-lining epithelial cells with rosiglitazone treatment. Analysis of cell cycle regulatory proteins revealed that rosiglitazone decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, cyclin D2 and Cdk4 but increased the levels of p21 and p27 in a dose-dependent manner. Rosiglitazone also induced apoptosis in cyst-lining epithelial cells, which was correlated with increased bax expression and decreased bcl-2 expression. These results suggest PPARgamma agonist might serve as a promising drug for the treatment of ADPKD.

  6. Cysteine modified polyaniline films improve biocompatibility for two cell lines.

    PubMed

    Yslas, Edith I; Cavallo, Pablo; Acevedo, Diego F; Barbero, César A; Rivarola, Viviana A

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using l-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV-visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86°±1 to 90°±1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering.

  7. Fibronectin synthesized by a human hepatoma cell line

    SciTech Connect

    Glasgow, J.E.; Colman, R.W.

    1984-07-01

    Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-(/sup 35/S)methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis.

  8. Chromosomal assignment of human DNA fingerprint sequences by simultaneous hybridization to arbitrarily primed PCR products from human/rodent monochromosome cell hybrids

    SciTech Connect

    Yasuda, Jun; Sekiya, Takao; Navarro, J.M.

    1996-05-15

    We have developed a technique for the simultaneous chromosomal assignment of multiple human DNA sequences from DNA fingerprints obtained by the arbitrarily primed polymerase chain reaction (AP-PCR). Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylong membrane. Human-specific hybridization bands in the human/rodent fingerprints unambiguously determine their chromosome of origin. We named this method simultaneous hybridization of arbitrarily primed PCR DNA fingerprinting products (SHARP). Using this approach, we determined the chromosomal origins of most major bands of human AP-PCR fingerprints obtained with two arbitrary primers. Altogether, the chromosomal localization of near 50 DNA fragments, comprehensive of all human chromosomes except chromosomes 21 and Y, was achieved in this simple manner. Chromosome assignment of fingerprint bands is essential for molecular karyotyping of cancer by AP-PCR DNA fingerprinting. The SHARP method provides a convenient and powerful tool for this purpose. 23 refs., 3 figs., 2 tabs.

  9. A preliminary study of side population cells in human gastric cancer cell line HGC-27.

    PubMed

    Gao, Ganglong; Sun, Zhenliang; Wenyong, Liu; Dongxia, Ye; Zhao, Runjia; Zhang, Xueli

    2015-03-16

    Cancer stem cell-like side population (SP) cells, which may be responsible for recurrence, tumor metastasis, and resistance to cancer therapy, have been identified and characterized in several types of cell lines from gastric cancer. However, there is no report on isolation of SP cells from human gastric cancer cell line HGC-27. This study aims to analyze the proportion of SP cells in HGC-27 cell line, differentiate SP from non-side population (NSP) cells, and determine whether the SP cells have certain biological properties of stem cells. (1) HGC-27 suspension was prepared and stained with Hoechst33342 and PI for flow cytometric isolation of SP (2). Differences in proliferation and stemness-related gene expression profiles (CD133, CD44, OCT-4, MDR1, EpCAM, and ABCG2) between SP and NSP cells were detected by gastric formation assay and quantitative real-time PCR (3). Oncogenicity of SP and NSP cells was determined in nude mice in vivo. (1) SP cells accounted for 0.1-1.0% of HGC-27 cells, and decreased to 0% after verapamil inhibition. Using flow cytometry, we sorted 7.5×10⁵ SP cells and most HGC-27 cells were NSP cells (2). Gastric formation assay and MTT demonstrated that there was a significant difference in proliferation between SP and NSP cells. Gene expression analysis showed that the expression of genes was significantly higher in SP cells (3). The oncogenicity experiment in nude mice revealed that 105 SP cells were able to form tumors, which demonstrated higher tumorigenicity than non-SP cells. These results collectively suggested that SP cells from HGC-27 cell line have some cancer stem cell properties and could be used for studying the pathogenesis of gastric cancer, which may contribute to discovery of novel therapeutic targets.

  10. The dual role of TLR3 in metastatic cell line.

    PubMed

    Matijevic, Tanja; Pavelic, Jasminka

    2011-10-01

    Toll-like receptors (TLRs) are members of transmembrane proteins that recognize conserved molecular motifs of viral and bacterial origin and initiate innate immune response. As the role of TLRs in tumors cells is still not clear, our aim was to investigate the role of TLR3 in primary tumor and metastatic cells (SW480, SW620, FaDu and Detroit 562). We have reported here on the dual role of TLR3 in pharynx metastatic cell line (Detroit 562); on one hand TLR3 activation drove cells to apoptosis while on the other its stimulation contributed to tumor progression by altering the expression of tumor promoting genes (PLAUR, RORB) and enhancing the cell migration potential. In addition, we have shown TLR3 signaling pathway is functional in another metastatic cancer cell line (SW620) suggesting TLR3 might be important in the process of tumor metastasis. Since TLR3 agonists have been used in tumor therapy with the aim to activate immune system, scientific contribution of this work is drawing attention to the importance of further work on this topic, especially pro-tumor effect of TLR3, in order to avoid possible side-effects.

  11. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  12. Role of glutathione in cisplatin resistance in osteosarcoma cell lines.

    PubMed

    Komiya, S; Gebhardt, M C; Mangham, D C; Inoue, A

    1998-01-01

    This study was designed to examine whether and how glutathione and catalase increase the resistance of osteosarcoma cells to the toxicity of cisplatin. Eight osteosarcoma cell lines were exposed to varying concentrations of cisplatin, and a [3H]thymidine incorporation study then estimated their drug sensitivity. Cells were pretreated with aminotriazole and buthionine sulfoximine to depress catalase and glutathione activities and then entered into the same protocol to assess their sensitivity to cisplatin. Intracytoplasmic levels of catalase and glutathione were measured before and after the treatments. Cisplatin-glutathione conjugates were created to examine how glutathione might depress the toxicity of cisplatin. Although the cell lines differed in the magnitude of their response to cisplatin, there was a statistical correlation between intrinsic glutathione content and cisplatin resistance. Pretreatment with aminotriazole reduced catalase activity by 84% but did not change the sensitivity to cisplatin. Depletion of glutathione activity by 70% increased the sensitivity of the cells to the cytotoxicity of cisplatin. In addition, cisplatin was detoxified following conjugation with glutathione. The increased sensitization to cisplatin toxicity caused by the depletion of glutathione and cisplatin detoxification after the in vitro reaction of glutathione to cisplatin indicated that the formation of the glutathione-cisplatin conjugate was an important mechanism in the cellular resistance to cisplatin. These data also demonstrated that catalase activity did not contribute to resistance to cisplatin and suggested that H2O2-induced oxidative stress did not significantly contribute to the cytotoxicity of cisplatin in osteosarcoma cells.

  13. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

    PubMed Central

    Biskup, Edyta; Manfé, Valentina; Kamstrup, Maria R.; Gniadecki, Robert

    2010-01-01

    We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa), Sézary syndrome (SeAx), and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK). Mac1 and Mac2a had the highest growth rate (doubling time 18–28 h, >90% cycling cells) whereas SeAx was proliferating slowly (doubling time 55 h, approximately 35% cycling cells). Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma. PMID:25386244

  14. Preclinical Analysis of Fetal Human Mesencephalic Neural Progenitor Cell Lines: Characterization and Safety In Vitro and In Vivo.

    PubMed

    Moon, Jisook; Schwarz, Sigrid C; Lee, Hyun-Seob; Kang, Jun Mo; Lee, Young-Eun; Kim, Bona; Sung, Mi-Young; Höglinger, Günter; Wegner, Florian; Kim, Jin Su; Chung, Hyung-Min; Chang, Sung Woon; Cha, Kwang Yul; Kim, Kwang-Soo; Schwarz, Johannes

    2016-09-02

    : We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening.

  15. Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.

    PubMed

    Stringfellow, David A; Riddell, Kay P; Givens, M Daniel; Galik, Patricia K; Sullivan, Eddie; Dykstra, Christine C; Robl, James; Kasinathan, Poothapillai

    2005-03-01

    Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

  16. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  17. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  18. Bisphosphonates induce apoptosis in human breast cancer cell lines

    PubMed Central

    Senaratne, S G; Pirianov, G; Mansi, J L; Arnett, T R; Colston, K W

    2000-01-01

    Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 μM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaign PMID:10780527

  19. Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes

    PubMed Central

    Truesdell, Sharon; Paul, Litty; Chen, Ting; Butchar, Jonathan P.; Justiniano, Steven

    2008-01-01

    Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype. PMID:18670627

  20. Antitumoral effect of vanadium compounds in malignant melanoma cell lines.

    PubMed

    Rozzo, Carla; Sanna, Daniele; Garribba, Eugenio; Serra, Maria; Cantara, Alessio; Palmieri, Giuseppe; Pisano, Marina

    2017-09-01

    In this study we evaluated the anticancer activity against malignant melanoma (MM) of four different vanadium species: the inorganic anion vanadate(V) (indicated with VN), and three oxidovanadium(IV) complexes, [V(IV)O(dhp)2] where dhp(-) is the anion 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS2), [V(IV)O(mpp)2] where mpp(-) is 1-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS3), and [V(IV)O(ppp)2] where ppp(-) is 1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS4). The antitumor effects of these compounds were studied against two different MM cell lines (A375 and CN-mel) and a fibroblast cell line (BJ) as normal control. All tested V compounds exert antiproliferative activity on MM cells in a dose dependent manner (IC50 ranges from 2.4μM up to 14μM) being A375 the most sensitive cell line. VN and VS2 were the two most active compounds against A375 (IC50 of 4.7 and 2.6μM, respectively), causing apoptosis and cell cycle block. The experimental data indicate that the cell cycle arrest occurs at different phases for the two V species analyzed (G2 checkpoint for VN and G0/G1 for VS2), showing the importance of the chemical form in determining their mechanism of action. These results add more insights into the landscape of vanadium versatility in biological systems and into its role as a potential cancer therapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Apoptosis induced by propolis in human hepatocellular carcinoma cell line.

    PubMed

    Choi, Y H; Lee, W Y; Nam, S Y; Choi, K C; Park, Y E

    1999-07-01

    Propolis has been reported to exhibit a wide spectrum of activities including antibiotic, antiviral, anti-inflammatory, immunostimulatory and tumor carcinostatic properties. We showed propolis induced apoptosis in a human hepatoma cell line (SNU449) by FITC-Annexin V/PI staining. We also compared the apoptosis inducing effect between Korean and Commercial (Sigma # p-1010) propolis. There was no difference on apoptosis between them.

  2. Characterization of stem-like cells in a new astroblastoma cell line.

    PubMed

    Coban, Esra Aydemir; Kasikci, Ezgi; Karatas, Omer Faruk; Suakar, Oznur; Kuskucu, Aysegul; Altunbek, Mine; Türe, Uğur; Sahin, Fikrettin; Bayrak, Omer Faruk

    2017-03-15

    Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Cholangiocarcinoma cell line TK may be useful for the pharmacokinetic study of the chemotherapeutic agent gemcitabine.

    PubMed

    Kamada, Minori; Akiyoshi, Kohei; Akiyama, Nobutake; Funamizu, Naotake; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Kei-Ichi; Manome, Yoshinobu

    2014-08-01

    Cholangiocarcinoma is a disease with a poor prognosis. A human cholangiocarcinoma cell line, TK, was previously established to enable further understanding of the disease. We conducted this investigation to determine whether or not the TK line is useful for pharmacokinetic study of the chemotherapeutic agent gemcitabine (GEM). Along with the BXPC3 human pancreatic adenocarcinoma cell line, the sensitivity to and effects on the TK cell line of GEM were compared. The influence of deoxycytidine kinase (dCK) transduction was also comparatively investigated. The effects of GEM in terms of drug sensitivity of the TK cell line, cell cycle and levels of transcripts of key enzymes were comparable to the BXPC3 cell line. Responses to the drug were similar in both cell lines. In contrast to pancreatic carcinoma, cell lines for research on cholangiocarcinoma have been limited. This study suggests the application of the TK cell line to the pharmacokinetic study of the chemosensitization of therapeutic drugs, such as GEM.

  4. Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines.

    PubMed

    Tomiya, Noboru; Narang, Someet; Lee, Yuan C; Betenbaugh, Michael J

    2004-01-01

    In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic

  5. Hepatitis C virus infection of cholangiocarcinoma cell lines.

    PubMed

    Fletcher, Nicola F; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K; van IJzendoorn, Sven C D; Baumert, Thomas F; Balfe, Peter; Afford, Simon; McKeating, Jane A

    2015-06-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo.

  6. Biotechnology and the chicken B cell line DT40.

    PubMed

    Bachl, J; Caldwell, R B; Buerstedde, J-M

    2007-01-01

    Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information (random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest within living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection. Copyright 2007 S. Karger AG, Basel.

  7. Effect of histone deacetylase inhibitor on proliferation of biliary tract cancer cell lines

    PubMed Central

    Xu, Li-Ning; Wang, Xin; Zou, Sheng-Quan

    2008-01-01

    AIM: To explore the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the growth of biliary tract cancer cell lines (gallbladder carcinoma cell line and cholangiocarcinoma cell line) in vivo and in vitro, and to investigate the perspective of histone deacetylase inhibitor in its clinical application. METHODS: The survival rates of gallbladder carcinoma cell line (Mz-ChA-l cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) treated with various doses of TSA were detected by methylthiazol tetrazolium (MTT) assay. A nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-l cell line) was successfully established, and changes in the growth of transplanted tumor after treated with TSA were measured. RESULTS: TSA could inhibit the proliferation of gallbladder carcinoma cell line (Mz-ChA-l cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) in a dose-dependent manner. After the nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-l cell line) was successfully established, the growth of cancer was inhibited in the model after treated with TSA. CONCLUSION: TSA can inhibit the growth of cholangiocarcinoma and gallbladder carcinoma cell lines in vitro and in vivo. PMID:18442209

  8. Identification and Characterization of CD133(pos) Subpopulation Cells From a Human Laryngeal Cancer Cell Line.

    PubMed

    Qiu, Hai-ou; Wang, Huifang; Che, Na; Li, Dong; Mao, Yong; Zeng, Qiao; Ge, Rongming

    2016-04-06

    Recent research indicates that CD133 are expressed in several kinds of stem cells, among which, its high expression in laryngeal carcinoma has caused wide concern. To further explore efficaciously targeting drugs to laryngeal carcinoma stem cells (CSCs), we transplanted a solid tumor from CSCs into abdominal subcutaneous tissue of nude mice, and then compared the biological characteristics of laryngeal solid tumors with or without cisplatin intervention. In this study, the expression of CD133 was detected in the Hep-2 cell line by flow cytometry. By applying magnetic cell sorting (MACS) technology, we reported the results of purifying CD133-positive cells from a Hep-2 cell line. Cell proliferation, colony formation, and tumor-forming ability were examined in vitro and in vivo to identify the marker of CSCs in Hep-2 cell line. Upon flow cytometry analysis, CD133 was expressed constantly on 40.12±1.32% in Hep-2 cell line. Cell proliferation and colony formation ability were higher in CD133-positive cells compared to CD133-negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay. The 2 subpopulations cells were both sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor-forming ability of CD133-positive cells was obviously greater than that of CD133-negative cells. Above all, our study revealed that CD133-positive cells have properties of higher proliferation, colony formation, and tumorigenesis in Hep-2 cell line, indicating that CD133 could be a marker to characterize laryngeal cancer stem cells.

  9. Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells

    PubMed Central

    Lawandi, Janet; Tao, Chang; Ren, Binhai; Williams, Paul; Ling, Dora; Swan, M Anne; Nassif, Najah T; Torpy, Fraser R; O’Brien, Bronwyn A; Simpson, Ann M

    2015-01-01

    As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D. PMID:26029722

  10. HIV-1 latency in actively dividing human T cell lines

    PubMed Central

    Jeeninga, Rienk E; Westerhout, Ellen M; van Gerven, Marja L; Berkhout, Ben

    2008-01-01

    Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFα treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus. PMID:18439275

  11. Biomarkers in Tumorigenesis Using Cancer Cell Lines: A Systematic Review

    PubMed

    Raju K, Lizbeth; Augustine, Dominic; Rao, Roopa S; S V, Sowmya; Haragannavar, Vanishri C; Nambiar, Shwetha; Prasad, Kavitha; Awan, Kamran Habib; Patil, Shankargouda

    2017-09-27

    Cancer is a leading cause of death worldwide. Despite many research advancements in the field, the genetic changes regulating the transformation of normal oral cells into malignant cells have not been fully elucidated. Several studies have evaluated carcinogenesis at the molecular level. Cancer cell lines are commonly used in biomedical research because they provide an unlimited source of cells and represent various stages of initiation and progression of carcinogenesis in vitro. Aims: The objective of the study was to review original research articles using cancer cell lines as a tool to understand carcinogenesis and to identify the genes involved in tumor development. Additionally, we also examined the application of the genes as predictive biomarkers. Methods and Materials: Several databases, including PubMed, Google Scholar, Ebsco, and Science Direct, were searched from 1985 to December 2016 using various combinations of the following key words: “mouth neoplasm”, “cell lines”, and “tumorigenesis”. Original experimental studies published in English were included. We excluded letters to the editor, historic reviews, and unpublished data from the analysis. Results: There were 17 studies (in vitro) included in the analysis. There were 14 genes and 4 miRNAs involved in malignant transformation of oral keratinocytes into cancer cells. The most commonly studied genes were p53, cyclin D1, and hTERT. Conclusion: Additional reviews and studies are needed to identify a panel of genes specific to various potentially malignant disorders and to aid in the early detection of oral squamous cell carcinoma (OSCC) because tumorigenesis involves the mutation of multiple genes. Furthermore, improving advanced cost-effective diagnostic methods may benefit the public health sector. Creative Commons Attribution License

  12. Bombesin stimulates insulin secretion by a pancreatic islet cell line.

    PubMed Central

    Swope, S L; Schonbrunn, A

    1984-01-01

    The amphibian tetradecapeptide, bombesin (BBS) has been shown to stimulate insulin secretion both in vivo and by pancreatic islet cells in vitro. To determine whether BBS can act directly on pancreatic beta cells, we examined its effects on insulin secretion by HIT-T15 cells (HIT cells), a clonal islet cell line. Addition of 100 nM BBS to HIT cells stimulated insulin release 25-fold within 30 sec. The rapid stimulatory effect of BBS on insulin release was short-lived: the secretory rate returned to basal levels after 90 min of BBS treatment. The decrease in the rate of insulin release in the continued presence of BBS was due not to depletion of intracellular insulin stores but to specific desensitization to this peptide. Stimulation of insulin secretion by BBS was dose dependent with an ED50 value (0.51 +/- 0.15 nM) similar to the concentration of BBS-like immunoreactive material in rat plasma. Five BBS analogs, including porcine gastrin-releasing peptide, were as powerful as BBS in stimulating insulin release. The relative potencies of the analogs tested indicated that the COOH-terminal octapeptide sequence in BBS was sufficient for stimulation of release. In contrast, 14 peptides structurally unrelated to BBS did not alter insulin secretion. BBS action was synergistic with that of glucagon; insulin secretion in the presence of maximal concentrations of both peptides was greater than the additive effects of the two peptides added individually. Somatostatin inhibited BBS-stimulated release by 69 +/- 1% with an ID50 value of 3.2 +/- 0.3 nM. These results show that BBS stimulation of insulin secretion by a clonal pancreatic cell line closely parallels its effects in vivo and support the hypothesis that BBS stimulates insulin secretion by a direct effect on the pancreatic beta cell. The clonal HIT cell line provides a homogeneous cell preparation amenable for studies on the biochemical mechanisms of BBS action in the endocrine pancreas. PMID:6143320

  13. Cell culture methods for the establishment of the NCI series of lung cancer cell lines.

    PubMed

    Oie, H K; Russell, E K; Carney, D N; Gazdar, A F

    1996-01-01

    More than 200 human small cell lung cancer and non-small cell lung cancer cell lines were established over 15 years mainly by utilizing the serum-free, hormone and growth factor supplemented, defined media HITES and ACL4. Use of modified, established cell culture techniques such as the mechanical spillout method for the releasing of cell aggregates from tumor tissue, ficoll gradient centrifugation for the separation of tumor cells from erythrocytes and tissue debris, and an apparatue consisting of a platinum tubing attached to a suction flask for removal of spent medium have greatly contributed to the success in culturing tumor cells. Characterization of these lung cancer cell lines have extended our knowledge of lung cell biology. Studies elucidating the nutritional requirements of lung cancer cell growth may be helpful for the manipulation of these tumors in patients.

  14. 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

  15. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. © 2016. Published by The Company of Biologists Ltd.

  16. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells

    PubMed Central

    Pujol, Remy; Cunningham, Dale E.; Hailey, Dale W.; Prendergast, Andrew; Rubel, Edwin W.; Raible, David W.

    2016-01-01

    ABSTRACT Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. PMID:27103160

  17. Cytogenetic instability of dental pulp stem cell lines.

    PubMed

    Duailibi, Monica Talarico; Kulikowski, Leslie Domenici; Duailibi, Silvio Eduardo; Lipay, Monica Vannucci Nunes; Melaragno, Maria Isabel; Ferreira, Lydia Masako; Vacanti, Joseph Phillip; Yelick, Pamela Crotty

    2012-02-01

    Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.

  18. [Mechanisms of gamma-inducible death of Jurkat cells line].

    PubMed

    Gamkrelidze, M M; Bezhitashvili, N D; Pavliashvili, A T; Mchedlishvili, T V; Sanikidze, T V

    2008-06-01

    Mechanisms of radio-inducible death of Jurkat cells were investigated. Human lymphoblastoid T-cell line Jurkat is widely established model for studying apoptosis mechanisms. The cell was radiated by "Teragam" (Czech Republic) by dose 2 g during 1 minute. After radiation cells were incubated at standard conditions during 24 hours. After gamma radiation in cell population amount of cells in gaplois (apoptotic G 0) stage was increased 8,2 folds, in diplois (G 0/G1) stage - by 17%, in synthetic (S) stage decreased by 35% and tetraploid (G2/M) stage by 73% in comparison to control group. It was revealed intensive production of free radicals of oxygen and nitric oxide and decreasing activity of antioxidant enzymes (superoxidismutasa, catalasa and glutathione peroxidase). Revealed dependence between intensification of apoptosis and radiation-induced arrest of cell cycle G2/M phase may be determined by excess amount of free oxygen and nitrogen radicals generated in Jurkat cells as a result of nondirect effects of low doses of gamma radiation.

  19. Inhibition of protease-resistant prion protein formation in a transformed deer cell line infected with chronic wasting disease.

    PubMed

    Raymond, Gregory J; Olsen, Emily A; Lee, Kil Sun; Raymond, Lynne D; Bryant, P Kruger; Baron, Gerald S; Caughey, Winslow S; Kocisko, David A; McHolland, Linda E; Favara, Cynthia; Langeveld, Jan P M; van Zijderveld, Fred G; Mayer, Richard T; Miller, Michael W; Williams, Elizabeth S; Caughey, Byron

    2006-01-01

    Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

  20. Pleomorphism and drug resistant cancer stem cells are characteristic of aggressive primary meningioma cell lines.

    PubMed

    Khan, Ishaq; Baeesa, Saleh; Bangash, Mohammed; Schulten, Hans-Juergen; Alghamdi, Fahad; Qashqari, Hanadi; Madkhali, Nawal; Carracedo, Angel; Saka, Mohamad; Jamal, Awatif; Al-Maghrabi, Jaudah; AlQahtani, Mohammed; Al-Karim, Saleh; Damanhouri, Ghazi; Saini, Kulvinder; Chaudhary, Adeel; Abuzenadah, Adel; Hussein, Deema

    2017-01-01

    Meningioma tumors arise in arachnoid membranes, and are the most reported central nervous system (CNS) tumors worldwide. Up to 20% of grade I meningioma tumors reoccur and currently predictive cancer stem cells (CSCs) markers for aggressive and drug resistant meningiomas are scarce. Meningioma tissues and primary cell lines were investigated using whole transcriptome microarray analysis, immunofluorescence staining of CSCs markers (including CD133, Sox2, Nestin, and Frizzled 9), and drug treatment with cisplatin or etoposide. Unsupervised hierarchical clustering of six meningioma samples separated tissues into two groups. Analysis identified stem cells related pathways to be differential between the two groups and indicated the de-regulation of the stem cell associated genes Reelin (RELN), Calbindin 1 (CALB1) and Anterior Gradient 2 Homolog (AGR2). Immunofluorescence staining for four tissues confirmed stemness variation in situ. Biological characterization of fifteen meningioma primary cell lines concordantly separated cells into two functionally distinct sub-groups. Pleomorphic cell lines (NG type) grew significantly faster than monomorphic cell lines (G type), had a higher number of cells that express Ki67, and were able to migrate aggressively in vitro. In addition, NG type cell lines had a lower expression of nuclear Caspase-3, and had a significantly higher number of CSCs co-positive for CD133+ Sox2+ or AGR2+ BMI1+. Importantly, these cells were more tolerant to cisplatin and etoposide treatment, showed a lower level of nuclear Caspase-3 in treated cells and harbored drug resistant CSCs. Collectively, analyses of tissues and primary cell lines revealed stem cell associated genes as potential targets for aggressive and drug resistant meningiomas.

  1. 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.

  2. Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

    PubMed

    Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

    2012-10-01

    Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches.

  3. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

    USDA-ARS?s Scientific Manuscript database

    The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and wer...

  4. Distinct differences in the responses of the human pancreatic β-cell line EndoC-βH1 and human islets to proinflammatory cytokines

    PubMed Central

    Oleson, Bryndon J.; McGraw, Jennifer A.; Broniowska, Katarzyna A.; Annamalai, Mani; Chen, Jing; Bushkofsky, Justin R.; Davis, Dawn B.; Mathews, Clayton E.

    2015-01-01

    While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling β-cell function and viability, translating findings to human β-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human β-cell line, EndoC-βH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-βH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-βH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-βH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-βH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-βH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-βH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-βH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-βH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line. PMID:26084699

  5. Distinct differences in the responses of the human pancreatic β-cell line EndoC-βH1 and human islets to proinflammatory cytokines.

    PubMed

    Oleson, Bryndon J; McGraw, Jennifer A; Broniowska, Katarzyna A; Annamalai, Mani; Chen, Jing; Bushkofsky, Justin R; Davis, Dawn B; Corbett, John A; Mathews, Clayton E

    2015-09-01

    While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling β-cell function and viability, translating findings to human β-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human β-cell line, EndoC-βH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-βH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-βH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-βH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-βH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-βH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-βH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-βH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-βH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line.

  6. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    PubMed

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent