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Sample records for gill cell line

  1. In vitro assay for the toxicity of silver nanoparticles using heart and gill cell lines of Catla catla and gill cell line of Labeo rohita.

    PubMed

    Taju, G; Abdul Majeed, S; Nambi, K S N; Sahul Hameed, A S

    2014-04-01

    Silver nanoparticles (Ag-NPs) are used in commercial products for their antimicrobial properties. The Ag-NPs in some of these products are likely to reach the aquatic environment, thereby posing a health concern for humans and aquatic species. The silver nanoparticles were synthesized and characterized using, UV-vis spectra, Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) analysis. Acute toxicity tests on fish were conducted by exposing Catla catla and Labeo rohita for 96h to AgNO3 and Ag-NPs under static conditions. The cytotoxic effect of AgNO3 and Ag-NPs in Sahul India C. catla heart cell line (SICH), Indian C. catla gill cell line (ICG) and L. rohita gill cell line (LRG) was assessed using MTT and neutral red (NR) assay. Linear correlations between each in vitro EC50 and the in vivo LC50 data were highly significant. DNA damage and nuclear fragmentation (condensation) were assessed by comet assay and Hoechst staining, respectively in SICH, ICG and LRG cells exposed to Ag-NPs. The results of antioxidant parameter obtained show significantly increased lipid peroxidation (LPO) level and decreased level of GSH, SOD and CAT in SICH, ICG and LRG cell lines after exposure to increasing Ag-NPs in a concentration-dependent manner. This work proves that fish cell lines could be used as an alternative to whole animals using cytotoxicity tests, genotoxicity tests and oxidative stress assessment after exposure to nanoparticles. PMID:24524868

  2. In vitro acute cytotoxicity of abamectin to the Gill Cell Line of Flounder Paralichthy olivaceus

    NASA Astrophysics Data System (ADS)

    Xu, Yuyan; Guo, Huarong; Xiao, Qin; Su, Feng; Yin, Licheng

    2007-10-01

    The cytotoxicity of abamectin to the Gill Cell Line of Flounder (FG cell line) was examined in this study. It was found that the exposure of FG cells to abamectin caused the decreases of both cell growth rate and antioxidant enzyme activities, and the increase of intracellular O2 - content. It was proposed that the reduction of antioxidant enzyme activities in FG cells caused the accumulation of O2 - content in FG cells, leading to the change of cell morphology and even the death of cells. The results showed that FG cell line is suitable for the evaluation of the acute toxicity of abamectin.

  3. In vitro study on cytotoxic effects of the organopho-sphorous pesticide profenofos on the gill cell line, FG-9307, of the flounder ( Paralichihys olivaceus)

    NASA Astrophysics Data System (ADS)

    Li, Hong-Yan; Zhang, Shi-Cui; Jiang, Ming; Wang, Min

    2001-03-01

    The FG-9007 cell line derived from the gill of flounder Paralichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that the cell growth rate was markedly reduced by profenofos at the concentrations of 2.5, 5 and 7.5 mg/L tested; and that the fine structures of the cells were also altered by profenofos, as evidenced by dilation of nuclear membranes and mitochondria cristae, and presence of enlarged lysosomes with engulfed organelles and numerous vacuoles in the cytoplasm. Probably, mitochondria, the cell energy-generating sites, are the most prominent sites of profenofos cytotoxity in the cells. This seems to be the first report of the use of marine fish cell line for evaluation of the acute in vitro cytotoxicity of organophosphorus pesticide.

  4. Development of the microsporidian parasite, Loma salmonae, in a rainbow trout gill epithelial cell line (RTG-1): evidence of xenoma development in vitro.

    PubMed

    McConnachie, S H; Sheppard, J; Wright, G M; Speare, D J

    2015-02-01

    Growth and propagation of fish-infecting microsporidians within cell culture has been more difficult to achieve than for insect- and human-infecting microsporidians. Fish microsporidia tend to elicit xenoma development rather than diffuse growth in vivo, and this process likely increases host specificity. We present evidence that the fish microsporidian, Loma salmonae, has the capacity to develop xenomas within a rainbow trout gill epithelial cell line (RTG-1). Spore numbers increased over a 4 weeks period within cell culture flasks. Xenoma-like structures were observed using phase contrast microscopy, and then confirmed using transmission electron microscopy. Optimization of the L. salmonae-RTG-1 cell model has important implications in elucidating the process of xenoma development induced by microsporidian parasites. PMID:25434457

  5. Cytotoxicity, genotoxicity and oxidative stress of malachite green on the kidney and gill cell lines of freshwater air breathing fish Channa striata.

    PubMed

    Majeed, S Abdul; Nambi, K S N; Taju, G; Vimal, S; Venkatesan, C; Hameed, A S Sahul

    2014-12-01

    The cytotoxicity, genotoxicity and oxidative stress of malachite green (MG) was investigated using the fish Channa striata kidney (CSK) and Channa striata gill (CSG) cell lines. Five concentrations ranging from 0.001 to 10 μg mL(-1) were tested in three independent experiments. Cytotoxicity was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Rhodamine 123 and Alamar Blue. The mitochondrial changes and apoptosis of MG-exposed cells were observed by Rhodamine 123 and acridine orange/ethidium bromide (AO/EB) staining, respectively. In vitro potential DNA damaging effect of MG was tested using comet assay. Mitochondrial damage, apoptosis and DNA fragmentation increased in a concentration-dependent manner. Additionally, DNA electrophoretic mobility experiments were carried out to study the binding effect of MG to double-stranded DNA (dsDNA) of cells. DNA shift mobility experiments showed that MG is capable of strongly binding to linear dsDNA causing its degradation. Biochemical parameters such as lipid peroxidation (MDA), catalase (CAT) activity and reduced glutathione (GSH) levels were evaluated after exposure to MG. In CSK and CSG cell lines exposed to MG for 48 h, a significant increase in lipid peroxidation, which might be associated with decreased levels of reduced glutathione and catalase activity in these cell lines (p < 0.001), was observed. PMID:25023653

  6. Surface Defects on Plate-Shaped Silver Nanoparticles Contribute to Its Hazard Potential in a Fish Gill Cell Line and Zebrafish Embyos

    PubMed Central

    George, Saji; Lin, Sijie; Ji, Zhaoxia; Thomas, Courtney; Li, LinJiang; Mecklenburg, Mathew; Meng, Huan; Wang, Xiang; Zhang, Haiyuan; Xia, Tian; Lin, Shuo; Hohman, J. Nathan; Zink, Jeffrey I.; Weiss, Paul; Nel, André E.

    2014-01-01

    We investigated and compared nano-size Ag spheres, plates, and wires in a fish gill epithelial cell line (RT-W1) and in zebrafish embryos to understand the mechanism of toxicity of an engineered nanomaterial raising considerable environmental concern. While most of the Ag nanoparticles induced N-acetyl cysteine sensitive toxic oxidative stress effects in RT-W1, Ag nanoplates were considerably more toxic than other particle shapes. Interestingly, while Ag ion shedding and bioavailability failed to explain the high toxicity of the nanoplates, cellular injury required direct particle contact, resulting in cell membrane lysis in RT-W1 as well as red blood cells (RBC). Ag nanoplates were also considerably more toxic in zebrafish embryos in spite of their lesser ability to shed Ag into the exposure medium. In order to elucidate the “surface reactivity” of Ag nanoplates, high-resolution transmission electron microscopy was performed and demonstrated a high level of crystal defects (stacking faults and point defects) on the nanoplate surfaces. Surface coating with cysteine was used to passivate the surface defects and demonstrated a reduction of toxicity in RT-W1 cells, RBC, and zebrafish embryos. This study demonstrates the important role of crystal defects in contributing to Ag nanoparticle toxicity in addition to the established roles of Ag ion shed from spherical nanoparticles. The excellent correlation between the in vitro and in vivo toxicological assessment illustrates the utility of using a fish cell line in parallel with zebrafish embryos to perform a predictive environmental toxicological paradigm. PMID:22482460

  7. Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line.

    PubMed

    Kühnel, Dana; Busch, Wibke; Meissner, Tobias; Springer, Armin; Potthoff, Annegret; Richter, Volkmar; Gelinsky, Michael; Scholz, Stefan; Schirmer, Kristin

    2009-06-28

    Due to their increased production and use, engineered nanoparticles are expected to be released into the aquatic environment where particles may agglomerate. The aim of this study was to explore the role of agglomeration of nanoparticles in the uptake and expression of toxicity in the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1. This cell line was chosen as model because it is known to be amenable to culture in complete as well as greatly simplified exposure media. Nano-sized tungsten carbide (WC) with or without cobalt doping (WC-Co), two materials relevant in the heavy metal industry, were applied as model particles. These particles were suspended in culture media with decreasing complexity from L15 with 10% fetal bovine serum (FBS) to L15 to L15/ex, containing only salts, galactose and pyruvate of the complete medium L15. Whereas the serum supplement in L15 retained primary nanoparticle suspensions, agglomerates were formed quickly in L15 and L15/ex. Nevertheless, scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) elemental analysis revealed an uptake of both WC and WC-Co nanoparticles into RTgill-W1 cells irrespective of the state of agglomeration of nanoparticles. The localisation seemed to be restricted to the cytoplasm, as no particles were observed in the nucleus of cells. Moreover, reduction in cell viability between 10 and 50% compared to controls were observed upon particle exposure in all media although the pattern of impact varied depending on the medium and exposure time. Short-term exposure of cells led to significant cytotoxicity at the highest nominal particle concentrations, irrespective of the particle type or exposure medium. In contrast, long-term exposures led to preferential toxicity in the simplest medium, L15/ex, and an enhanced toxicity by the cobalt-containing WC nanoparticles in all exposure media. The composition of the exposure media also influenced the toxicity of the cobalt ions, which may

  8. Impact of detergents on the protein histochemistry of various cell types of the gill epithelium of Rita rita.

    PubMed

    Roy, D

    1988-04-01

    Fish, Rita rita, were exposed to an anionic detergent, dodecylbenzene sodium sulfonate, 6.9 mg per litre of tap water (96-hr LC50 of the detergent). A gradual decrease in the protein constituents of the major cell types, viz, the epithelial cells and the goblet mucous cells in the epithelium lining the gill arch, gill filament, and club cells present only in the gill arch epithelium has been observed by using a series of histochemical techniques. PMID:3378537

  9. Cell Lines

    PubMed Central

    Cherbas, Lucy; Gong, Lei

    2014-01-01

    We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals. PMID:24434506

  10. Cell signaling and ion transport across the fish gill epithelium.

    PubMed

    Evans, David H

    2002-08-01

    A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in

  11. Gill cell culture systems as models for aquatic environmental monitoring.

    PubMed

    Bury, Nic R; Schnell, Sabine; Hogstrand, Christer

    2014-03-01

    A vast number of chemicals require environmental safety assessments for market authorisation. To ensure acceptable water quality, effluents and natural waters are monitored for their potential harmful effects. Tests for market authorisation and environmental monitoring usually involve the use of large numbers of organisms and, for ethical, cost and logistic reasons, there is a drive to develop alternative methods that can predict toxicity to fish without the need to expose any animals. There is therefore a great interest in the potential to use cultured fish cells in chemical toxicity testing. This review summarises the advances made in the area and focuses in particular on a system of cultured fish gill cells grown into an epithelium that permits direct treatment with water samples. PMID:24574380

  12. Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium.

    PubMed

    Yue, Yang; Behra, Renata; Sigg, Laura; Schirmer, Kristin

    2016-10-01

    While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle-cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell-environment interface, offering novel opportunities to study nanoparticle-cell interactions without serum protein interference. PMID:27030289

  13. Trout gill cells in primary culture on solid and permeable supports.

    PubMed

    Leguen, I; Cauty, C; Odjo, N; Corlu, A; Prunet, P

    2007-12-01

    Trout gill cells in primary culture on solid and permeable supports were compared. Cultures were carried out by directly seeding cells on each support after gill dissociation. Most of the cell types present in culture were similar, regardless of culture support (pavement cells, mucous cells (3-4%), but no mitochondria-rich cells). However, insertion of mucous cells in cultured epithelium on permeable support presented a morphology more similar to gills in situ. Gene expression of ion transporters and hormonal receptors indicated similar mRNA levels in both systems. Cortisol inhibited cell proliferation on both supports and maintained or increased the total cell number on solid and permeable membranes, respectively. This inhibition of mitosis associated with an increase or maintenance of total gill cells suggests that cortisol reduced cell degeneration. In the presence of cortisol, transepithelial resistance of cultured gill cells on permeable membranes was increased and maintained for a longer time in culture. In conclusion, gill cells in primary culture on permeable support present: (i) a morphology more similar to epithelium in situ; and (ii) specific responses to cortisol treatment. New findings and differences with previous studies on primary cultures of trout gill cells on permeable membrane are discussed. PMID:17977040

  14. Statistical analysis of anionic detergent-induced changes in the goblet mucous cells of opercular epidermis and gill epithelium of Rita rita (Ham.) (Bagridae: Pisces).

    PubMed

    Roy, D

    1988-06-01

    Rita rita exposed to 96-hr LC50 (6.9 mg/liter) of an anionic detergent, dodecylbenzene sodium sulfonate, show significant changes in the number and size of goblet mucous cells in the opercular epidermis as well as in the lining epithelium of the gill arch and the gill filament at different time intervals of treatment. A shift in the staining nature of these cells from acidic glycoprotein to neutral glycoprotein, acidic glycosaminoglycans in the opercular epidermis and acidic glycoprotein to neutral glycoprotein and then again to acidic glycoprotein + acidic glycosaminoglycans in the gill filament epithelium reflects a change in the physiological status of fish. PMID:3168874

  15. Characterization of copper transport in gill cells of a mangrove crab Ucides cordatus.

    PubMed

    Sá, M G; Zanotto, F P

    2013-11-15

    The branchial epithelium of crustaceans is exposed to the environment and is the first site affected by metal pollution. The aim of this work was to characterize copper (Cu) transport using a fluorescent dye, Phen Green, in gill cells of a hypo-hyper-regulator mangrove crab Ucides cordatus. The results showed that added extracellular CuCl2 (0, 0.025, 0.150, 0.275, 0.550 and 1.110 μM) showed typical Michaelis-Menten transport for Cu in anterior and posterior gill cells (Vmax for anterior and posterior gills: 0.41 ± 0.12 and 1.76 ± 0.27 intracellular Cu in μM × 22.10(4)cells(-1)× 300 s(-1) respectively and Km values: 0.44 ± 0.04 and 0.32 ± 0.13 μM, respectively). Intracellular Cu was significantly higher for posterior gill cells compared to anterior gill cells, suggesting differential accumulation for each gill type. Extracellular Ca at 20mM decreased cellular Cu transport for both anterior and posterior gill cells. Nifedipine and verapamil, calcium channel inhibitors from plasma membrane, decreased Cu transport and affected Km for both gills. These results could be due to a competition between Cu and Ca. Amiloride, a Na/Ca exchanger inhibitor, as well as bafilomycin, a proton pump inhibitor, caused a decrease of intracellular Cu compared to control. Ouabain and KB-R 7943, acting on Na homeostasis, similarly decreased intracellular Cu in both gill cells. Besides that, gill cells exposed to ATP and Cu simultaneously, showed an increase in intracellular copper, which was inhibited by vanadate, an inhibitor of P-type ATPase. These results suggest either the presence of a Cu-ATPase in crab gill cells, responsible for Cu influx, or the effect of a change in electrochemical membrane potential that could also drive Cu to the gill cell interior. Caffeine increased intracellular Cu, suggesting that intracellular Ca could be affecting Cu uptake. Overall the results show that copper uptake in gill cells of crabs is regulated by intracellular Ca, Ca channels and by Na

  16. 65Zn2+ transport by isolated gill epithelial cells of the American lobster, Homarus americanus.

    PubMed

    Sá, M G; Ahearn, G A; Zanotto, F P

    2009-07-01

    Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in 65Zn2+ in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 muM verapamil, nifedipine, and the calcium ionophore A23187. 65Zn2+ influxes were hyperbolic functions of zinc concentration (1-1,000 microM) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. 65Zn2+ transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented. PMID:19198852

  17. In vitro effect of various xenobiotics on trout gill cell volume regulation after hypotonic shock.

    PubMed

    Leguen, I; Prunet, P

    2001-08-01

    Their functions and localisation can expose gill cells to volume changes. To maintain their vital functions, these gill cells must regulate their own volume after cellular swelling or shrinkage. Recently, we showed that rainbow trout pavement gill cells in primary culture have the capacity to regulate their own volume after cellular swelling induced by hypotonic shock. This so-called regulatory volume decrease (RVD) is associated with intracellular calcium increase, which occurs as a transient peak followed by a plateau when maintained a hypotonic condition. Return to an isotonic medium restores baseline [Ca2+]i level. In this study, the effect of different xenobiotics on cellular swelling induced RVD and its calcium signal was investigated in trout pavement gill cells in primary culture. These cells were exposed to different pollutants after confluent epithelium was obtained. After 36 h in xenobiotics exposure in vitro, cellular volume and intracellular calcium concentration were measured. Nonylphenol poly- and di-ethoxylate were lethal at concentrations of 10 and 100 microM, respectively. With 10 microM of the diethoxylate form, cells did not die but, unlike non-treated cells, burst during hypotonic shock (2/3rd strength Ringer solution). With 1 microM nonylphenol polyethoxylate (NPnEO), RVD and [Ca2+]i were reduced. Copper (10 and 100 microM) had no significant effect on gill cell volume regulation. However, the heavy metal modified calcium response to hypotonic shock by inhibiting return to baseline level under isotonic conditions. 10 microM prochloraz and 2,4-dichloroaniline had no effect on cell morphology, volume and [Ca2+]i concentration. With 100 microM, however, prochloraz was lethal and dichloroaniline increased baseline [Ca2+]i. These results indicate that the effects observed on gill cells are consistent with the known toxic properties of the molecules tested, thus confirming the validity of primary culture to investigate the toxic effects of

  18. Toxic effects of an anionic detergent on the lipid constituents of various cell types of the gill epithelium of Rita rita: a histochemical investigation.

    PubMed

    Roy, D

    1989-12-01

    Rita rita exposed to a concentration of 6.9 mg per liter (96-h LC50 of an anionic detergent, dodecylbenzene sodium sulfonate) exhibited a gradual decrease in the lipid moieties of the epithelial cells, club cells, and goblet mucous cells lining the gill arch and gill filament epithelium. However, in time, no reaction of any of the lipid moieties could be observed, indicating the absence of the same, using various histochemical techniques. The results are discussed in light of the mechanistic understanding of detergent action. PMID:2604897

  19. Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture.

    PubMed

    Leguen; Carlsson; Perdu-Durand; Prunet; Pärt; Cravedi

    2000-03-01

    The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10(-6) to 10(-12) M. After 24 h, TCDD showed a maximal induction at a concentration of 10(-9) M while BNF showed a maximal induction at a concentration of 10(-7) M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 µM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6beta and 16beta and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate. PMID:10686323

  20. Natural Mineral Particles Are Cytotoxic to Rainbow Trout Gill Epithelial Cells In Vitro

    PubMed Central

    de Capitani, Christian; Burkhardt-Holm, Patricia; Pietsch, Constanze

    2014-01-01

    Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L−1) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H2DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8–1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6–1.8-fold-changes at the 250 mg L−1 concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3–1.6-fold increases at the 250 mg L−1 concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species

  1. Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei.

    PubMed

    Granado e Sá, Marina; Baptista, B B; Farah, L S; Leite, V P; Zanotto, F P

    2010-03-01

    Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca(2+)) transport because of molting-related events and the deposition of CaCO(3) in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca(2+) transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca(2+) change was monitored by adding Ca as CaCl(2) (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca(2+) transport followed Michaelis-Menten kinetics with K(m) = 0.42 +/- 0.04 mM and V(max) = 0.50 +/- 0.02 microM (Ca(2+) change x initial intracellular Ca(-1) x 180 s(-1); N = 14, r (2) = 0.99). Verapamil (a Ca(2+) channel inhibitor) and amiloride (a Na(+)/Ca(2+) exchanger [NCX] inhibitor) completely reduced intracellular Ca(2+) transport, while nifedipine, another Ca(2+) channel inhibitor, did not. Vanadate, a plasma membrane Ca(2+)-ATPase inhibitor (PMCA), increased intracellular Ca(2+) in gill cells through a decrease in the efflux of Ca(2+). Ouabain increased intracellular Ca(2+), similar to the effect of KB-R, a specific NCX inhibitor for Ca(2+) in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca(2+) transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca(2+) compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca(2+) in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca(2+) homeostasis through various Ca(2+) intracellular sequestration mechanisms and/or plasma membrane Ca(2+) influx

  2. Impact of low dose of organophosphate, monocrotophos on the epithelial cells of gills of Cyprinus carpio communis Linn.--SEM study.

    PubMed

    Johal, M S; Sharma, M L; Ravneet

    2007-07-01

    The paper deals with the deleterious changes at ultrastructural level of the epithelial cells of gills of Cyprinus carpio communis Linn. upon exposure to 1/10th of LC50 of monocrotophos which is considered to be insignificant concentration from toxicological point of view. The gills of the fish are the primary corridor formolecularexchange between the internal milieu of a fish and its environment. Gills perform numerous functions such as oxygen uptake and CO2 excretion, osmoregulation, acid-basic balance, excretion of nitrogenous compounds and taste. Hazardous chemicals present in water may alter the morphology of the epithelial cells of gills of the fish, which may affect the process of diffusion of gases and ultimately the overall health of the fish. To prove this fact Cyprinus carpio communis Linn. was kept in water for 30 days having low concentration of 0.038 ppm (1\\10th of LC50) of monocrotophos and an attempt was made to study the different types of degenerations produced in the epithelial cells of gills as compared to the normal epithelial cells of gills of this culturable fish using Scanning Electron Microscope (SEM) technique. The ultrastructural changes due to the toxic exposure at finer scale were thinning of microridges, upliftment of epithelial cells, development of hyperplasia, decrease in the density of mucous cells which are considered to be the first line of defence and total dystrophy of epithelial tissue. Thus, it is opined that a low concentration of monocrotophos has the potential to bring different type of degenerations at finer scale hence affecting the fish's health drastically and altering the fitness of the fish in water even having insignificant amount of this toxicant in the ambient water PMID:18380092

  3. Calcium transport in gill cells of Ucides cordatus, a mangrove crab living in variable salinity environments.

    PubMed

    Leite, V P; Zanotto, F P

    2013-10-01

    Crustaceans show discontinuous growth and have been used as a model system for studying cellular mechanisms of calcium transport, which is the main mineral found in their exoskeleton. Ucides cordatus, a mangrove crab, is naturally exposed to fluctuations in calcium and salinity. To study calcium transport in this species during isosmotic conditions, dissociated gill cells were marked with fluo-3 and intracellular Ca(2+) change was followed by adding extracellular Ca(2+) as CaCl2 (0, 0.1, 0.25, 0.50, 1.0 and 5mM), together with different inhibitors. For control gill cells, Ca(2+) transport followed Michaelis-Menten kinetics with Vmax=0.137±0.001 ∆Ca(2+)i (μM×22.10(4)cells(-1)×180s(-1); N=4; r(2)=0.99); Km=0.989±0.027mM. The use of different inhibitors for gill cells showed that amiloride (Na(+)/Ca(2+) exchange inhibitor) inhibited 80% of Ca(2+) transport in gill cells (Vmax). KB-R, an inhibitor of Ca influx in vertebrates, similarly caused a decrease in Ca(2+) transport and verapamil (Ca(2+) channel inhibitor) had no effect on Ca(2+) transport, while nifedipine (another Ca(2+) channel inhibitor) caused a 20% decrease in Ca(2+) affinity compared to control values. Ouabain, on the other hand, caused no change in Ca(2+) transport, while vanadate increased the concentration of intracellular calcium through inhibition of Ca(2+) efflux probably through the plasma membrane Ca(2+)-ATPase. Results show that transport kinetics for Ca(2+) in these crabs under isosmotic conditions is lower compared to a hyper-regulator freshwater crab Dilocarcinus pagei studied earlier using fluorescent Ca(2+) probes. These kinds of studies will help understanding the comparative mechanisms underlying the evolution of Ca transport in crabs living in different environments. PMID:23867752

  4. Gills of antarctic fish.

    PubMed

    Rankin, J C; Tuurala, H

    1998-01-01

    We review the literature on the way the structure of icefish gills relates the physiology of these haemoglobin-less fishes. Vascular casting confirmed earlier reports that the only special feature of the gills is the large size of the blood vessels, especially the prominent and continuous marginal channels Isolated perfused gill arches were used to study the effects of changes in afferent and efferent pressure on gill resistance and tritiated water influx in Chionobathyscus dewitti. Increasing perfusion rate did not change gill resistance, but there were moderate proportional increases in water influx. Reducing efferent pressure increased gill resistance but did not affect water influx. In both C. dewitti and Cryodraco antarcticus gills perfused at constant flow rate, noradrenaline produced concentration-dependent decreases in gill resistance and, with high concentrations, increases in water influx. Fixation while perfusion continued was used to compare blood space dimensions in control, noradrenaline-treated and unperfused gills. Noradrenaline caused large increases in the thickness of the lamellar blood space and increased lamellar height, despite a greatly reduced afferent pressure. This suggests that modulation of pillar cell active tension might be involved in control of lamellar perfusion. The possible relationship between gill water fluxes and lamellar recruitment is discussed. PMID:11253779

  5. Innervation of Gill Lateral Cells in the Bivalve Mollusc Crassostrea virginica Affects Cellular Membrane Potential and Cilia Activity

    PubMed Central

    Catapane, Edward J; Nelson, Michael; Adams, Trevon; Carroll, Margaret A

    2016-01-01

    Gill lateral cells of Crassostrea virginica are innervated by the branchial nerve, which contains serotonergic and dopaminergic fibers that regulate cilia beating rate. Terminal release of serotonin or dopamine results in an increase or decrease, respectively, of cilia beating rate in lateral gill cells. In this study we used the voltage sensitive fluorescent probe DiBAC4(3) to quantify changes in gill lateral cell membrane potential in response to electrical stimulation of the branchial nerve or to applications of serotonin and dopamine, and correlate these changes to cilia beating rates. Application of serotonin to gill lateral cells caused prolonged membrane depolarization, similar to plateau potentials, while increasing cilia beating rate. Application of dopamine hyperpolarized the resting membrane while decreasing cilia beating rate. Low frequency (5 Hz) electrical stimulations of the branchial nerve, which cause terminal release of endogenous serotonin, or high frequency (20 Hz) stimulations, which cause terminal release of endogenous dopamine, had the same effects on gill lateral cell membrane potentials and cilia beating rate as the respective applications of serotonin or dopamine. The study shows that innervation of gill lateral cells by the branchial nerve affects membrane potential as well as cilia beating rate, and demonstrates a strong correlation between changes in membrane potential and regulation of cilia beating rate. The study furthers the understanding of serotonin and dopamine signaling in the innervation and regulation of gill cilia in bivalves. The study also shows that voltage sensitive fluorescent probes like DiBAC 4(3) can be successfully used as an alternative to microelectrodes to measure changes in membrane potential of ciliated gill cells and other small cells with fast moving cilia. PMID:27489887

  6. Detection of 8-oxodG in Dreissena polymorpha gill cells exposed to model contaminants.

    PubMed

    Michel, Cécile; Vincent-Hubert, Françoise

    2012-01-24

    Genotoxic end-points are routinely measured in various sentinel organisms in aquatic environments in order to monitor the impact of water pollution on organisms. As a first step towards the evaluation of oxidative DNA damage (8-oxodG) in organisms exposed to chemical water pollution, we have optimized the association between the comet assay and the hOGG1 enzyme for use on zebra mussel (Dreissena polymorpha) gill cells by in vitro exposure to H₂O₂. Firstly, we observed that in vitro exposure of D. polymorpha gill cells to benzo[a]pyrene (B[a]P, 98.4nM) induced an increase of the Olive Tail Moment (OTM) in both the comet-hOGG1 and comet-Fpg assays, indicating that B[a]P causes oxidative DNA damage. By contrast, methylmethane sulfonate (MMS, 33μM) only induced an increase of the Fpg-sensitive sites, indicating that MMS caused alkylating DNA damage and confirming that hOGG1 does not detect alkylating damage. Thus, the hOGG1 enzyme seems to be more specific towards oxidative DNA damage, such as 8-oxodG than Fpg. Secondly, as was observed in vitro, the in vivo exposure of D. polymorpha to B[a]P (24.6 and 98.4nM) increased oxidative DNA damage in gill cells, whereas only Fpg-sensitive sites were detected in mussels exposed to MMS (240μM). These results show that the comet-hOGG1 assay detects oxidative DNA lesions induced in vitro by H₂O₂ and in vivo with BaP. The comet-hOGG1 assay will be used to detect oxidative DNA lesions (8-oxodG) in mussels exposed in situ. PMID:22009068

  7. In situ detection of apoptotic cells by TUNEL in the gill epithelium of the developing brown trout (Salmo trutta)

    PubMed Central

    ROJO, M. C.; GONZALEZ, M. E.

    1998-01-01

    Apoptosis is a form of naturally occurring cell death during development and it is characterised by extensive DNA fragmentation. Apoptosis is easily detected in the gill epithelium of brown trout embryos in ultrathin sections (Rojo et al. 1997). Here we provide the first biochemical evidence for apoptosis in the gill epithelium of brown trout embryos, using in situ end-labelling of DNA breaks (Gavrieli et al. 1992). Embryos at d 57 of development as well as those at hatching, were processed to analyse the distribution of apoptotic cells in the gills. The extent of apoptosis revealed by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labelling method technique is considerably greater than apoptosis detected by nuclear morphology. This method revealed that apoptosis was frequent at hatching, although it was also present during embryonic development. The presence and distribution of stained nuclei were different depending on the developmental stage. In embryos of 57 d, apoptotic flattened nuclei were dispersed in the gill epithelium, whereas at hatching, they were mainly grouped in the tips of the filaments and in the gill arches. TUNEL also revealed a distinct pattern of nuclear staining: at hatching, the intense staining covered the entire cell, but in embryos it was restricted to the nucleus. These results show the functional relevance of apoptosis at hatching, when apoptosis seems to be the unique process by which cell numbers in the gill epithelium are adjusted, in order to prepare for the new extrinsic conditions affecting the free-living life of alevins. PMID:9877294

  8. Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation.

    PubMed

    Kültz, D; Somero, G N

    1996-01-01

    Different protein patterns in gill epithelium of a euryhaline and eurythermal teleost fish (Gillichthys mirabilis, Family Gobiidae) in response to long-term (2 months) osmotic and thermal acclimation were found for the first time. Gill epithelial cells were isolated to remove extracellular proteins and quantify specialized cell types. Chloride cells were identified on the basis of size (> 10 microns) and bright appearance after [2-(p-dimethylaminostyryl)-1-methyl-pyridinium-iodine] staining. Small mitochondria-rich cells were < 5 microns in diameter and showed intermediate fluorescence. Abundance of chloride cells and small mitochondria-rich cells was significantly influenced by osmotic but not thermal acclimation (dilute seawater/25 degrees C: 1.4 +/- 0.2% chloride cells, 11.9 +/- 4.6% small mitochondria-rich cells; seawater/25 degrees C: 2.4 +/- 0.6% chloride cells, 2.2 +/- 1.3% small mitochondria-rich cells; seawater/10 degrees C: 2.9 +/- 0.3% chloride cells, 1.2 +/- 0.7% small mitochondria-rich cells). Pavement cells, identified by low fluorescence and intermediate size (5-10 microns), largely predominated under all conditions (> 85% of cells). Thus, they represented the major protein source in gill epithelium. Differences in protein patterns were detectable using two-dimensional but not one-dimensional electrophoresis. Of 602 proteins identified by charge and molecular weight properties, only two were induced by high temperature (25 degrees C) and three in response to cold acclimation (10 degrees C). Nine proteins were induced in diluted seawater-acclimated fish, whereas no seawater-induced proteins were found. We hypothesize that proteins induced under dilute seawater conditions are important for the function of pavement cells in gills of hyper-osmoregulating G. mirabilis. PMID:8766907

  9. Light microscopic characterization of glycoconjugates in secretory cells of the carp (Cyprinus carpio) gill epithelium.

    PubMed

    Hidalgo, J; Velasco, A; Sánchez Aguayo, I; Amores, P

    1987-01-01

    Secretory products of granular and mucous cells in the gill epithelium of the carp, Cyprinus carpio, were distinguished by their cytochemical reactions with peroxidase-labelled lectins and with the galactose oxidase (GO)-Schiff reagents. Secretory products of granular cells reacted with lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Glycine max (SAB), and Lotus tetragonolobus (LTA). They also reacted with GO-Schiff reagents. After sialic acid cleavage with HCl, new binding sites for DBA and SBA appeared, suggesting the terminal sequence sialic acid-N-acetylgalactosamine (SA-GalNAc) for the secretion of this cell type. In mucous cells, binding sites for WGA, DBA, and SBA and, after acid hydrolysis, binding sites for PNA and a positive GO-Schiff reaction were detected. The terminal trisaccharide sialic acid-galactose (beta 1-3)-N-acetylgalactosamine (SA-Gal-GalNAc) is proposed for the secretion of mucous cells. These cytochemical differences are discussed in light of the involvement of both cell types in fish mucus elaboration. PMID:2449406

  10. Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

    PubMed

    Al-Hussinee, L; Pham, P H; Russell, S; Tubbs, L; Tafalla, C; Bols, N C; Dixon, B; Lumsden, J S

    2016-09-01

    The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression. PMID:26850791

  11. A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: evidence for passive and facilitated transport.

    PubMed

    Stott, Lucy C; Schnell, Sabine; Hogstrand, Christer; Owen, Stewart F; Bury, Nic R

    2015-02-01

    The gill is the principle site of xenobiotic transfer to and from the aqueous environment. To replace, refine or reduce (3Rs) the large numbers of fish used in in vivo uptake studies an effective in vitro screen is required that mimics the function of the teleost gill. This study uses a rainbow trout (Oncorhynchus mykiss) primary gill cell culture system grown on permeable inserts, which tolerates apical freshwater thus mimicking the intact organ, to assess the uptake and efflux of pharmaceuticals across the gill. Bidirectional transport studies in media of seven pharmaceuticals (propranolol, metoprolol, atenolol, formoterol, terbutaline, ranitidine and imipramine) showed they were transported transcellularly across the epithelium. However, studies conducted in water showed enhanced uptake of propranolol, ranitidine and imipramine. Concentration-equilibrated conditions without a concentration gradient suggested that a proportion of the uptake of propranolol and imipramine is via a carrier-mediated process. Further study using propranolol showed that its transport is pH-dependent and at very low environmentally relevant concentrations (ng L(-1)), transport deviated from linearity. At higher concentrations, passive uptake dominated. Known inhibitors of drug transport proteins; cimetidine, MK571, cyclosporine A and quinidine inhibited propranolol uptake, whilst amantadine and verapamil were without effect. Together this suggests the involvement of specific members of SLC and ABC drug transporter families in pharmaceutical transport. PMID:25544062

  12. A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: Evidence for passive and facilitated transport

    PubMed Central

    Stott, Lucy C.; Schnell, Sabine; Hogstrand, Christer; Owen, Stewart F.; Bury, Nic R.

    2015-01-01

    The gill is the principle site of xenobiotic transfer to and from the aqueous environment. To replace, refine or reduce (3Rs) the large numbers of fish used in in vivo uptake studies an effective in vitro screen is required that mimics the function of the teleost gill. This study uses a rainbow trout (Oncorhynchus mykiss) primary gill cell culture system grown on permeable inserts, which tolerates apical freshwater thus mimicking the intact organ, to assess the uptake and efflux of pharmaceuticals across the gill. Bidirectional transport studies in media of seven pharmaceuticals (propranolol, metoprolol, atenolol, formoterol, terbutaline, ranitidine and imipramine) showed they were transported transcellularly across the epithelium. However, studies conducted in water showed enhanced uptake of propranolol, ranitidine and imipramine. Concentration-equilibrated conditions without a concentration gradient suggested that a proportion of the uptake of propranolol and imipramine is via a carrier-mediated process. Further study using propranolol showed that its transport is pH-dependent and at very low environmentally relevant concentrations (ng L−1), transport deviated from linearity. At higher concentrations, passive uptake dominated. Known inhibitors of drug transport proteins; cimetidine, MK571, cyclosporine A and quinidine inhibited propranolol uptake, whilst amantadine and verapamil were without effect. Together this suggests the involvement of specific members of SLC and ABC drug transporter families in pharmaceutical transport. PMID:25544062

  13. Blasted Cell Line Names

    PubMed Central

    Wang, Jing; Byers, Lauren A.; Yordy, John S.; Liu, Wenbin; Shen, Li; Baggerly, Keith A.; Giri, Uma; Myers, Jeffrey N.; Ang, K. Kian; Story, Michael D.; Girard, Luc; Minna, John D.; Heymach, John V.; Coombes, Kevin R.

    2010-01-01

    Background: While trying to integrate multiple data sets collected by different researchers, we noticed that the sample names were frequently entered inconsistently. Most of the variations appeared to involve punctuation, white space, or their absence, at the juncture between alphabetic and numeric portions of the cell line name. Results: Reasoning that the variant names could be described in terms of mutations or deletions of character strings, we implemented a simple version of the Needleman-Wunsch global sequence alignment algorithm and applied it to the cell line names. All correct matches were found by this procedure. Incorrect matches only occured when a cell line was present in one data set but not in the other. The raw match scores tended to be substantially worse for the incorrect matches. Conclusions: A simple application of the Needleman-Wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets. PMID:21082038

  14. Distinct freshwater and seawater isoforms of Na+/K+-ATPase in gill chloride cells of Atlantic salmon

    USGS Publications Warehouse

    McCormick, Stephen D.; Regish, A.M.; Christensen, A.K.

    2009-01-01

    Gill Na(+)/K(+)-ATPase (NKA) in teleost fishes is involved in ion regulation in both freshwater and seawater. We have developed and validated rabbit polyclonal antibodies specific to the NKA alpha1a and alpha1b protein isoforms of Atlantic salmon (Salmo salar Linnaeus), and used western blots and immunohistochemistry to characterize their size, abundance and localization. The relative molecular mass of NKA alpha1a is slightly less than that for NKA beta1b. The abundance of gill NKA alpha1a was high in freshwater and became nearly undetectable after seawater acclimation. NKA alpha1b was present in small amounts in freshwater and increased 13-fold after seawater acclimation. Both NKA isoforms were detected only in chloride cells. NKA alpha1a was located in both filamental and lamellar chloride cells in freshwater, whereas in seawater it was present only as a faint background in filamental chloride cells. In freshwater, NKA alpha1b was found in a small number of filamental chloride cells, and after seawater acclimation it was found in all chloride cells on the filament and lamellae. Double simultaneous immunofluorescence indicated that NKA alpha1a and alpha1b are located in different chloride cells in freshwater. In many chloride cells in seawater, NKA alpha1b was present in greater amounts in the subapical region than elsewhere in the cell. The combined patterns in abundance and immunolocalization of these two isoforms can explain the salinity-related changes in total NKA and chloride cell abundance. The results indicate that there is a freshwater and a seawater isoform of NKA alpha-subunit in the gills of Atlantic salmon and that they are present in distinct chloride cells.

  15. Mast cells in the intestine and gills of the sea bream, Sparus aurata, exposed to a polychlorinated biphenyl, PCB 126.

    PubMed

    Lauriano, Eugenia Rita; Calò, Margherita; Silvestri, Giuseppa; Zaccone, Daniele; Pergolizzi, Simona; Lo Cascio, Patrizia

    2012-02-01

    The presence of mast cells has been reported in all classes of vertebrates, including many teleost fish families. The mast cells of teleosts, both morphologically and functionally, show a close similarity to the mast cells of mammals. Mast cells of teleosts, localized in the vicinity of blood vessels of the intestine, gills and skin, may play an important role in the mechanisms of inflammatory response, because they express a number of functional proteins, including piscidins, which are antimicrobical peptides that act against a broad-spectrum of pathogens. An increase in the number of mast cells in various tissues and organs of teleosts seems to be linked to a wide range of stressful conditions, such as exposure to heavy metals (cadmium, copper, lead and mercury), exposure to herbicides and parasitic infections. This study analyzed the morphological localization and abundance of mast cells in the intestine and gills of sea bream, Sparus aurata, after a 12, 24 or 72 h exposure to PCB 126, a polychlorinated biphenyl, which is a potent immunotoxic agent. In the organs of fish exposed to PCB 126, it was observed that in addition to congestion of blood vessels, there was extravasation of red blood cells, infiltration of lymphocytes, and a progressive increase in numbers of mast cells. These data confirm the immunotoxic action of PCB, and the involvement of mast cells in the inflammatory response. PMID:21565388

  16. 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

  17. Environmental monitoring of urban streams using a primary fish gill cell culture system (FIGCS).

    PubMed

    Schnell, Sabine; Bawa-Allah, Kafilat; Otitoloju, Adebayo; Hogstrand, Christer; Miller, Thomas H; Barron, Leon P; Bury, Nic R

    2015-10-01

    The primary fish gill cell culture system (FIGCS) is an in vitro technique which has the potential to replace animals in whole effluent toxicity tests. In the current study FIGCS were transported into the field and exposed to filtered (0.2μm) river water for 24h from 4 sites, on 2 different sampling dates. Sites 1 and 2 are situated in an urban catchment (River Wandle, London, UK) with site 1 downstream of a sewage treatment work; site 3 is located in a suburban park (River Cray, Kent, UK), and site 4 is more rural (River Darent, Kent, UK). The change in transepithelial electrical resistance (TER), the expression of the metal responsive genes metallothionein A (mta) and B (mtb), cytochrome P450 1A1 (cyp1a1) and 3A27 (cyp3a27), involved in phase 1 metabolism, were assessed following exposure to sample water for 24h. TER was comparable between FIGCS exposed to 0.2μm filtered river water and those exposed to synthetic moderately soft water for 24h. During the first sampling time, there was an increase in mta, cyp1a1 and cyp3a27 gene expression in epithelium exposed to water from sites 1 and 2, and during the second sampling period an increase in cyp3a27 gene expression at sites 1 and 4. Urban river water is a complex mixture of contaminants (e.g., metals, pesticides, pharmaceuticals and polyaromatic hydrocarbons) and the increase in the expression of genes encoding mta, cyp1a1 and cyp3a27 in FIGCS is indicative of the presence of biologically active pollutants. PMID:26093110

  18. Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics.

    PubMed

    Smith, Richard W; Wood, Chris M; Cash, Phil; Diao, Linda; Pärt, Peter

    2005-05-20

    The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 microg ml(-1) human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function. PMID:15848139

  19. Primary cultured cells as sensitive in vitro model for assessment of toxicants--comparison to hepatocytes and gill epithelia.

    PubMed

    Zhou, Bingsheng; Liu, Chunsheng; Wang, Jingxian; Lam, Paul K S; Wu, Rudolf S S

    2006-11-16

    In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3h, and was maximal after 6h of exposure to TCDD, B[a]P and Aroclor 1254. The estimated 6h EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.2 x 10(-9), 5.7 x 10(-8) and 6.6 x 10(-6)M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24h after exposure. The estimated 24h EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4 x 10(-9), 8.1 x 10(-8) and 7.3 x 10(-6)M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants. PMID:16959333

  20. DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis during anoxic stress and aerobic recovery

    NASA Astrophysics Data System (ADS)

    Slobodskova, Valentina V.; Zhukovskaya, Avianna F.; Chelomin, Victor P.

    2012-06-01

    Anoxia-induced DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis was assessed with the alkaline comet assay (single-cell gel electrophoresis). The alkaline comet assay method for detecting DNA strand breaks and alkali labile sites in individual cells. DNA damage was determened in the scallops ( M. yessoensis) gill cells. The scallops were exposed to air for 8 h showing a clear increase in the levels of DNA damage. After the air exposure, M. yessoensis were re-submersed for a period of 12 h, leading values to return to a pre-aerial exposure level. Control animals were kept immersed during the whole period. The resulting data demonstrate that natural influences, such as oxygen depletion (anoxia) in seawater, can be responsible for the induction of DNA damage. If the scallops were re-immersed in oxic conditions, the anoxically induced breaks were repaired. The main mechanisms influencing the integrity of the DNA structure are discussed in this paper.

  1. Feeding induces translocation of vacuolar proton ATPase and pendrin to the membrane of leopard shark (Triakis semifasciata) mitochondrion-rich gill cells.

    PubMed

    Roa, Jinae N; Munévar, Christian L; Tresguerres, Martin

    2014-08-01

    In this study we characterized mitochondrion-rich (MR) cells and regulation of acid/base (A/B) relevant ion-transporting proteins in leopard shark (Triakis semifasciata) gills. Immunohistochemistry revealed that leopard shark gills posses two separate cell populations that abundantly express either Na⁺/K⁺-ATPase (NKA) or V-H⁺-ATPase (VHA), but not both ATPases together. Co-immunolocalization with mitochondrial Complex IV demonstrated, for the first time in shark gills, that both NKA- and VHA-rich cells are also MR cells, and that all MR cells are either NKA- or VHA-rich cells. Additionally we localized the anion exchanger pendrin to VHA-rich cells, but not NKA-rich cells. In starved sharks, VHA was localized throughout the cell cytoplasm and pendrin was present at the apical pole (but not in the membrane). However, in a significant number of gill cells from fed leopard sharks, VHA translocated to the basolateral membrane (as previously described in dogfish), and pendrin translocated to the apical membrane. Our results highlight the importance of translocation of ion-transporting proteins to the cell membrane as a regulatory mechanism for A/B regulation. PMID:24746982

  2. Mechanisms of Toxicity of Ag Nanoparticles in Comparison to Bulk and Ionic Ag on Mussel Hemocytes and Gill Cells

    PubMed Central

    Katsumiti, Alberto; Gilliland, Douglas; Arostegui, Inmaculada; Cajaraville, Miren P.

    2015-01-01

    Silver nanoparticles (Ag NPs) are increasingly used in many products and are expected to end up in the aquatic environment. Mussels have been proposed as marine model species to evaluate NP toxicity in vitro. The objective of this work was to assess the mechanisms of toxicity of Ag NPs on mussel hemocytes and gill cells, in comparison to ionic and bulk Ag. Firstly, cytotoxicity of commercial and maltose stabilized Ag NPs was screened in parallel with the ionic and bulk forms at a wide range of concentrations in isolated mussel cells using cell viability assays. Toxicity of maltose alone was also tested. LC50 values were calculated and the most toxic Ag NPs tested were selected for a second step where sublethal concentrations of each Ag form were tested using a wide array of mechanistic tests in both cell types. Maltose-stabilized Ag NPs showed size-dependent cytotoxicity, smaller (20 nm) NPs being more toxic than larger (40 and 100 nm) NPs. Maltose alone provoked minor effects on cell viability. Ionic Ag was the most cytotoxic Ag form tested whereas bulk Ag showed similar cytotoxicity to the commercial Ag NPs. Main mechanisms of action of Ag NPs involved oxidative stress and genotoxicity in the two cell types, activation of lysosomal AcP activity, disruption of actin cytoskeleton and stimulation of phagocytosis in hemocytes and increase of MXR transport activity and inhibition of Na-K-ATPase in gill cells. Similar effects were observed after exposure to ionic and bulk Ag in the two cell types, although generally effects were more marked for the ionic form. In conclusion, results suggest that most observed responses were due at least in part to dissolved Ag. PMID:26061169

  3. Early genotoxic effects in gill cells and haemocytes of Dreissena polymorpha exposed to cadmium, B[a]P and a combination of B[a]P and Cd.

    PubMed

    Vincent-Hubert, Françoise; Arini, Adeline; Gourlay-Francé, Catherine

    2011-07-14

    The aim of this study was to assess the genotoxic potential of environmentally relevant concentrations of Cd on the zebra mussel, an important freshwater sentinel organism, and to determine the stability of DNA damage in gill cells and haemocytes. The oxidative DNA damage and the co-genotoxicity of Cd in combination with B[a]P were investigated. We measured DNA damage in haemocytes and gill cells of zebra mussels exposed for 11 days to a constant concentration of Cd (10μg/L), B[a]P (10μg/L) or the two combined chemicals (10μg/L+1μg/L). Enzymatic dissociation of gills with dispase gave the lower percentage DNA in tail, compared with collagenase/dispase or collagenase. Bioaccumulation of cadmium in the soft tissues of mussels exposed to CdCl(2) or CdCl(2)+B[a]P increased in a time-dependent manner indicating that both exposures were effective. Cd (10μg/L) is genotoxic only during the first 3 days of exposure in gill cells, while in haemocytes the genotoxicity of Cd was observed later. B[a]P (10μg/L) induced an early increase of DNA damage in gill cells (after 10h and 1 day), while in both gill cells and haemocytes, B[a]P caused a marked increase of DNA damage after 3 days of exposure. The Cd+B[a]P mixture decreased the DNA-damaging effect of Cd and B[a]P in both cell types. Cd induced an increase of DNA damage in Fpg-treated slides, indicating that Cd contributed to oxidative DNA damage. Cadmium induced a cytogenetic effect in gill cells, assessed by the number of micronuclei, throughout the duration of the exposure, while B[a]P did not induce any cytogenetic effect. B[a]P, Cd and Cd+B[a]P induced a transient increase in the number of bi-nucleated cells. Our data clearly show that gills are more sensitive to Cd and B[a]P, which makes them more suitable for future bio-monitoring studies. PMID:21453782

  4. Strain variability in fatty acid composition of Chattonella marina (Raphidophyceae) and its relation to differing ichthyotoxicity toward rainbow trout gill cells.

    PubMed

    Dorantes-Aranda, Juan José; Nichols, Peter D; David Waite, Trevor; Hallegraeff, Gustaaf M

    2013-04-01

    Lipid profiles of three strains (Mexico, Australia, Japan) of Chattonella marina (Subrahmanyan) Hara et Chihara were studied under defined growth (phosphate, light, and growth phase) and harvest (intact and ruptured cells) conditions. Triacylglycerol levels were always <2%, sterols <7%, free fatty acids varied between 2 and 33%, and polar lipids were the most abundant lipid class (>51% of total lipids). The major fatty acids in C. marina were palmitic (16:0), eicosapentaenoic (EPA, 20:5ω3), octadecatetraenoic (18:4ω3), myristic (14:0), and palmitoleic (16:1ω7c) acids. Higher levels of EPA were found in ruptured cells (21.4-29.4%) compared to intact cells (8.5-25.3%). In general, Japanese N-118 C. marina was the highest producer of EPA (14.3-29.4%), and Mexican CMCV-1 the lowest producer (7.9-27.1%). Algal cultures, free fatty acids from C. marina, and the two aldehydes 2E,4E-decadienal and 2E,4E-heptadienal (suspected fatty acid-derived products) were tested against the rainbow trout fish gill cell line RTgill-W1. The configuration of fatty acids plays an important role in ichthyotoxicity. Free fatty acid fractions, obtained by base saponification of total lipids from C. marina showed a potent toxicity toward gill cells (median lethal concentration, LC50 (at 1 h) of 0.44 μg · mL(-1) in light conditions, with a complete loss of viability at >3.2 μg · mL(-1) ). Live cultures of Mexican C. marina were less toxic than Japanese and Australian strains. This difference could be related to differing EPA content, superoxide anion production, and cell fragility. The aldehydes 2E,4E-decadienal and 2E,4E-heptadienal also showed high impact on gill cell viability, with LC50 (at 1 h) of 0.34 and 0.36 μg · mL(-1) , respectively. Superoxide anion production was highest in Australian strain CMPL01, followed by Japanese N-118 and Mexican CMCV-1 strains. Ruptured cells showed higher production of superoxide anion compared to intact cells (e.g., 19 vs. 9.5 pmol · cell

  5. Structural lipid changes and Na(+)/K(+)-ATPase activity of gill cells' basolateral membranes during saltwater acclimation in sea lamprey (Petromyzon marinus, L.) juveniles.

    PubMed

    Lança, Maria João; Machado, Maria; Ferreira, Ana Filipa; Quintella, Bernardo Ruivo; de Almeida, Pedro Raposo

    2015-11-01

    Seawater acclimation is a critical period for anadromous species and a process yet to be understood in lampreys. Considering that changes in lipid composition of the gill cells' basolateral membranes may disrupt the major transporter Na(+)K(+)-ATPase, the goal of this study was to detect changes at this level during juvenile sea lamprey seawater acclimation. The results showed that saltwater acclimation has a direct effect on the fatty acid composition of gill cells basolateral membrane's phospholipids. When held in full-strength seawater, the fatty acid profile of basolateral membrane's phospholipids suffered a restructure by increasing either saturation or the ratio between oleic acid and eicosapentaenoic acid. Simultaneously, the activity of Na(+)K(+)-ATPase revealed a significant and positive correlation with basolateral membrane's cholesterol content in the presence of highest salinity. Our results pointed out for lipid adjustments involving the functional transporter present on the gill cell basolateral membranes to ensure the role played by branchial Na(+)K(+)-ATPase in ion transport during saltwater acclimation process. The responses observed contributed to the strategy adopted by gill cell's basolateral membranes to compensate for osmotic and ionic stressors, to ensure the success of the process of seawater acclimation associated with the downstream trophic migration of juvenile sea lamprey. PMID:26244517

  6. Gill histopathology of Maria-da-toca Hypleurochilus fissicornis by metacercariae of Bucephalus margaritae (Digenea: Bucephalidae).

    PubMed

    Silva, Renato Z; da Costa Marchiori, Natalia; Magalhães, Aimê Rachel M; Cousin, João Carlos B; Romano, Luis Alberto; Pereira, Joaber

    2016-06-01

    Gills of Maria-da-toca Hypleurochilus fissicornis collected at Ponta do Sambaqui-Florianópolis island-Brazil, were analyzed to describe the histopathology caused by metacercaria of Bucephalus margaritae. Gills were submitted to the routine histological techniques for embedding in paraffin and permanent mounting in Balsam and stereoscopic analysis. Metacercariae showed a branchial infection site pattern for encystations. The branchial infection site pattern is half-basalward in the primary branchial filament with amplitude of the infection of 1-3 metacercaria. Cysts occurred within branchial abductor muscle and cartilaginous and osseous tissues of the gills. Each metacercariae had a contentional hyaline parasitic capsule and melanin-like pigmentation. The half-apicalward region of the primary branchial filaments showed several dysplasia degrees, cartilage and osseous degeneration (pyknosis), thrombosis and immune exudated cells (mainly lymphocytes). Cytopathologies as thickening of the epithelium lining of the secondary branchial filaments were a response of the branchial infection site pattern of the metacercaria. Interlamellar obliteration and fusion of the lamellae due to the hypertrophy and hyperplasia of the epithelial lining as well as chloride cells occurred. Pyknosis of pillar cells and epithelial lining cells from the secondary branchial filaments were also present. Bucephalosis in H. fissicornis gills is no-hemorrhagic and no-fatal branchitis, but could compromises the gill functions and could permits the secondary opportunistic infections. PMID:27413297

  7. 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. PMID:1726925

  8. Evaluation of cytotoxicity and genotoxicity of insecticide carbaryl to flounder gill cells and its teratogenicity to zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pandey, Manish Raj; Guo, Huarong

    2015-04-01

    In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill (FG) cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was determined with methods including MTT and neutral red uptaking (NRU), lactate dehydrogenase (LDH) releasing and Hoechst 33342 and propidium idodide (PI) double staining. Moderate cytotoxicity in a concentration-dependent manner was observed. The 24 h-IC50 value of 53.48 ± 1.21, 59.13 ± 1.19 and 46.21 ± 1.24 mg L-1 carbaryl was obtained through MTT, NRU and LDH assays, respectively. Double fluorescence staining demonstrated that carbaryl induced the death of FG cells mainly through necrosis. There was no significant genotoxicity found in the FG cells exposed to the highest testing concentration of carbaryl (20 mg L-1, P > 0.05) as was demonstrated by Comet assay. Zebrafish embryos exposed to carbaryl at concentrations ≥10 mg L-1 displayed moderate toxic effects on the survival, spontaneous movement, hatching, heart rates of the embryos and their development, which were evidenced by yolk and pericardial sac edemas, body length reduction and tail flexure in time- and concentration-dependent manners at specific stages. The 24 h-, 48 h- and 96 h-LC50 values of carbaryl to zebrafish embryos were 41.80 ± 1.10, 17.80 ± 1.04 and 14.46 ± 1.05 mg L-1, respectively. These results suggested that carbaryl is moderately toxic to FG cells cultured in vitro and zebrafish embryos, and the FG cells were similar to zebrafish embryos in their sensitivity to carbaryl as 24 h-IC50 and LC50 indicated.

  9. Differential impacts of elevated CO2 and acidosis on the energy budget of gill and liver cells from Atlantic cod, Gadus morhua.

    PubMed

    Stapp, L S; Kreiss, C M; Pörtner, H O; Lannig, G

    2015-09-01

    Ocean acidification impacts fish and other marine species through increased seawater PCO2 levels (hypercapnia). Knowledge of the physiological mechanisms mediating effects in various tissues of fish is incomplete. Here we tested the effects of extracellular hypercapnia and acidosis on energy metabolism of gill and liver cells of Atlantic cod. Exposure media mimicked blood conditions in vivo, either during normo- or hypercapnia and at control or acidic extracellular pH (pHe). We determined metabolic rate and energy expenditure for protein biosynthesis, Na(+)/K(+)-ATPase and H(+)-ATPase and considered nutrition status by measurements of metabolic rate and protein biosynthesis in media with and without free amino acids (FAA). Addition of FAA stimulated hepatic but not branchial oxygen consumption. Normo- and hypercapnic acidosis as well as hypercapnia at control pHe depressed metabolic stimulation of hepatocytes. In gill cells, acidosis depressed respiration independent of PCO2 and FAA levels. For both cell types, depressed respiration was not correlated with the same reduction in energy allocated to protein biosynthesis or Na(+)/K(+)-ATPase. Hepatic energy expenditure for protein synthesis and Na(+)/K(+)-ATPase was even elevated at acidic compared to control pHe suggesting increased costs for ion regulation and cellular reorganization. Hypercapnia at control pHe strongly reduced oxygen demand of branchial Na(+)/K(+)-ATPase with a similar trend for H(+)-ATPase. We conclude that extracellular acidosis triggers metabolic depression in gill and metabolically stimulated liver cells. Additionally, hypercapnia itself seems to limit capacities for metabolic usage of amino acids in liver cells while it decreases the use and costs of ion regulatory ATPases in gill cells. PMID:26005104

  10. Cortisol affects tight junction morphology between pavement cells of rainbow trout gills in single-seeded insert culture.

    PubMed

    Sandbichler, Adolf Michael; Farkas, Julia; Salvenmoser, Willi; Pelster, Bernd

    2011-12-01

    A primary culture system of rainbow trout gill pavement cells grown on permeable support (single-seeded insert, SSI) was used to examine histological and physiological changes induced by the addition of the corticosteroid hormone cortisol. Pavement cell epithelia were cultured under symmetrical conditions (L15 apical/L15 basolateral) and developed a high transepithelial resistance (TER, 6.84 ± 1.99 kΩ cm(2), mean ± SEM) with a low phenol red diffusion rate (PRD, 0.15 ± 0.03 μmol l(-1)/day). Addition of cortisol to the basolateral compartment increased TER twofold and reduced PRD threefold over a 5-day period. A similar increase in TER could be seen after 24 h apical freshwater (FW) in control cultures. In cortisol-treated cultures FW exposure did not change TER, but PRD increased significantly. Histochemical staining of the cytoskeleton of cells in SSI culture revealed a morphological partitioning into a single mucosal layer of polarized, polygonal cells featuring cortical F-actin rings which were comparable to F-actin rings of epithelial cells on the lamellar and filamental surface, and several unorganized serosal layers of cells with F-actin stress fibers. Addition of cortisol increased cell density by 18% and in the mucosal layer it led to smaller, less polygonal cells with increased height and increased cell contact area. In transmission electron microscopic images two pairs of cytoplasmatic electron-dense structures confining the zonula occludens apically and basally toward the zonula adhaerens were found. Addition of cortisol increased the distance between those paired structures, hence led to deeper tight junctions. The cortisol-induced increase in barrier properties, therefore, involves a structural fortification of the tight junctions which was not generally modified by a short 24-h apical freshwater stress. These results identify cortisol as a regulator of tight junction morphology between pavement cells of euryhaline fish such as the

  11. Effects of chronic exposure to soft, acidic water on gill development and chloride cell numbers in embryo-larval brook trout, Salvelinus fontinalis

    USGS Publications Warehouse

    Conklin, D.J.; Mowbray, R.C.; Gingerich, W.H.

    1992-01-01

    Recruitment failure is considered to be a major factor contributing to the decline of fish populations in soft, acidic waters; direct mortality of embryo-larval fishes has been postulated as a major cause of the decline. Little is understood of the physiological consequences to embryo-larval fishes of prolonged exposure to soft, acidic waters; however, dysfunction of respiratory and ionoregulatory processes is suspected. In order to evaluate the effects of acid exposure on the respiratory and ionoregulatory systems of developing brook trout, Salvelinus fontinalis, differences in gill morphology and numbers of chloride cells were compared between groups cf developing embryo-larval fish continuously exposed to moderately hard well water (130.0 mg.l-1 as CaCO3, pH 7.94) or to reconstituted soft, acidic water (4.4 mg.l-1 as CaCO3, pH 5.25) designed to mimic acidic waters of northern Wisconsin acidified lakes. Exposures were maintained for up to 48 days (82 days after fertilization) during critical periods of growth and differentiation of branchial structures. The second right gill arch of each fish was examined for changes in the development of filaments and lamellae and for differences in numbers of chloride cells. Gills of fish that developed in soft, acidic water contained greater numbers of normal and degenerating chloride cells, exhibited hyperplasia of primary epithelium and multiple fusions of adjacent filaments and lamellar epithelium than the gills of control fish. Filament and lamellar lengths and numbers of lamellae per filament were significantly less (P< 0.05) in fish that developed in soft, acidic water than in fish exposed to well water.

  12. The effect of pH on the toxicity of fatty acids and fatty acid amides to rainbow trout gill cells.

    PubMed

    Bertin, Matthew J; Voronca, Delia C; Chapman, Robert W; Moeller, Peter D R

    2014-01-01

    Harmful algal blooms (HABs) expose aquatic organisms to multiple physical and chemical stressors during an acute time period. Algal toxins themselves may be altered by water chemistry parameters affecting their bioavailability and resultant toxicity. The purpose of this study was to determine the effects of two abiotic parameters (pH, inorganic metal salts) on the toxicity of fatty acid amides and fatty acids, two classes of lipids produced by harmful algae, including the golden alga, Prymnesium parvum, that are toxic to aquatic organisms. Rainbow trout gill cells were used as a model of the fish gill and exposed to single compounds and mixtures of compounds along with variations in pH level and concentration of inorganic metal salts. We employed artificial neural networks (ANNs) and standard ANOVA statistical analysis to examine and predict the effects of these abiotic parameters on the toxicity of fatty acid amides and fatty acids. Our results demonstrate that increasing pH levels increases the toxicity of fatty acid amides and inhibits the toxicity of fatty acids. This phenomenon is reversed at lower pH levels. Exposing gill cells to complex mixtures of chemical factors resulted in dramatic increases in toxicity compared to tests of single compounds for both the fatty acid amides and fatty acids. These findings highlight the potential of physicochemical factors to affect the toxicity of chemicals released during algal blooms and demonstrate drastic differences in the effect of pH on fatty acid amides and fatty acids. PMID:24240104

  13. Adaptive alterations on gill Na⁺, K⁺-ATPase activity and mitochondrion-rich cells of juvenile Acipenser sinensis acclimated to brackish water.

    PubMed

    Zhao, Feng; Wu, Beibei; Yang, Gang; Zhang, Tao; Zhuang, Ping

    2016-04-01

    Understanding the physiological changes and osmoregulatory strategy is critical for anadromous species to adapt to large changes between freshwater and marine environments. In this study, juvenile Chinese sturgeon (Acipenser sinensis) were acclimated for 2 months to freshwater (FW, c. 0‰) and brackish water (BW, 15‰). Blood was assessed for changes in osmolality and ions. Gill tissue was assayed for Na(+), K(+)-ATPase (NKA) activity and immunohistochemical analysis on mitochondria-rich cells (MRCs). Serum osmolality and ions concentrations (Na(+), Cl(-) and K(+)) examined, except K(+), increased significantly in those specimens adapted to BW. However, the variations were within the range of effective hyperosmotic adaptation. The specific activity of gill NKA of juveniles adapted to BW was significantly higher (c. 1.6 times) than that of fish adapted to FW. MRCs were mainly presented in the interlamellar region of the filament and at the base of the lamella in either FW- or BW-acclimated individuals. In BW, the number and size of MRCs on filaments greatly increased. However, there was no significant difference in the number and size of the MRCs at the lamella region. Results show that juvenile Chinese sturgeon keep osmotic homeostasis in hyperosmotic environments by increasing gill NKA activity and MRCs' size and number, which is similar to other sturgeons and euryhaline teleosts. PMID:26614501

  14. Microtubule-dependent changes in morphology and localization of chloride transport proteins in gill mitochondria-rich cells of the tilapia, Oreochromis mossambicus.

    PubMed

    Yang, Wen-Kai; Wu, Yu-Ching; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-08-01

    The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na(+) /Cl(-) cotransporter (NCC) and Na(+) /K(+) /2Cl(-) cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria-rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time-course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule-dependent cellular regulating processes was used. SW-acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine-FW) for 96 h. Compared with the FW-treatment group, in the MR cells of colchicine-FW-treatment group, (1) the average size was significantly larger, (2) only wavy-convex-subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule-dependent. J. Morphol. 277:1113-1122, 2016. © 2016 Wiley Periodicals, Inc. PMID:27239784

  15. 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. PMID:24357037

  16. Human Adrenocortical Carcinoma Cell Lines

    PubMed Central

    Wang, Tao; Rainey, William E.

    2011-01-01

    Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies. PMID:21924324

  17. 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

  18. The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments.

    PubMed

    Kang, Chao-Kai; Yang, Wen-Kai; Lin, Shang-Tao; Liu, Chin-Cheng; Lin, Huei-Ming; Chen, Hong-Huan; Cheng, Chien-Wen; Lee, Tsung-Han; Hwang, Pung-Pung

    2013-01-01

    The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl(-) through the transporters including the Na(+), K(+)-ATPase (NKA), Na(+), K(+), 2Cl(-) cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl(-) secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (>1day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24h, the Cl(-) secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments. PMID:22960413

  19. The ichthyotoxic alga Chattonella marina induces Na{sup +}, K{sup +}-ATPase, and CFTR proteins expression in fish gill chloride cells in vivo

    SciTech Connect

    Tang, Janet Y.M.; Wong, Chris K.C.; Au, Doris W.T. . E-mail: bhdwtau@cityu.edu.hk

    2007-02-02

    Our previous studies demonstrated that the ichthyotoxic Chattonella marina stimulated proliferation of branchial chloride cell (CC) and induced osmotic distress akin to hyperactive elimination of ions in fish (Rhabdosargus sarba). To ascertain the in vivo effects of C. marina on key CC ion transporters, the localization and expression of Na{sup +}, K{sup +}-ATPase (NKA) and cystic fibrosis transmembrane conductance regulator (CFTR) proteins in response to C. marina exposure were investigated, using a quantitative immunocytochemical approach. The polarized distributions of NKA ({alpha} subunit) and CFTR proteins in branchial CCs of R. sarba remained unchanged under C. marina exposure. However, significant inductions of these two ion-transporters were detected in CCs of fish after 6 h exposure. By real-time PCR, no significant changes in gill NKA and CFTR mRNA expressions were detected, suggesting a post-transcriptional pathway is likely involved in regulating the ion transporters abundance. This study is the first to demonstrate the in vivo effects of harmful algal toxin on NKA and CFTR protein expressions in gill transepithelial cells. Taken together, an augmentation of branchial CCs together with hyper-stimulation of NKA and CFTR in CCs attribute to the rapid development of osmotic distress in C. marina susceptible fish.

  20. Diverse cell-specific expression of myoglobin isoforms in brain, kidney, gill and liver of the hypoxia-tolerant carp and zebrafish.

    PubMed

    Cossins, Andrew R; Williams, Daryl R; Foulkes, Nick S; Berenbrink, Michael; Kipar, Anja

    2009-03-01

    Myoglobin (Mb) is famous as a muscle-specific protein--yet the common carp expresses the gene (cMb1) encoding this protein in a range of non-muscle tissues and also expresses a novel isoform (cMb2) in the brain. Using a homologous antibody and riboprobes, we have established the relative amounts and cellular sites of non-muscle Mb expression in different tissues. The amounts of carp myoglobin (cMb) in supernatants of different tissues were just 0.4-0.7% relative to that of heart supernatants and were upregulated by two-to-four fold in liver, gill and brain following 5 days of hypoxic treatment. Brain exhibited both cMb proteins in western analysis, whereas all other tissues had only cMb1. We have also identified cells expressing cMb protein and cMb mRNA using immunohistology and RNA in situ hybridisation (RNA-ISH), respectively. Mb was strongly expressed throughout all cardiac myocytes and a subset of skeletal muscle fibres, whereas it was restricted to a small range of specific cell types in each of the non-muscle tissues. These include pillar and epithelial cells in secondary gill lamellae, hepatocytes, some neurones, and tubular epithelial cells in the kidney. Capillaries and small blood vessels in all tissues exhibited Mb expression within vascular endothelial cells. The cMb2 riboprobe located expression to a subset of neurones but not to endothelial cells. In zebrafish, which possesses only one Mb gene, a similar expression pattern of Mb protein and mRNA was observed. This establishes a surprisingly cell-specific distribution of Mb within non-muscle tissues in both carp and zebrafish, where it probably plays an important role in the regulation of microvascular, renal and brain function. PMID:19218513

  1. Claudin 28b and F-actin are involved in rainbow trout gill pavement cell tight junction remodeling under osmotic stress.

    PubMed

    Sandbichler, Adolf Michael; Egg, Margit; Schwerte, Thorsten; Pelster, Bernd

    2011-05-01

    Permeability of rainbow trout gill pavement cells cultured on permeable supports (single seeded inserts) changes upon exposure to freshwater or treatment with cortisol. The molecular components of this change are largely unknown, but tight junctions that regulate the paracellular pathway are prime candidates in this adaptational process. Using differential display polymerase chain reaction we found a set of 17 differentially regulated genes in trout pavement cells that had been exposed to freshwater apically for 24 h. Five genes were related to the cell-cell contact. One of these genes was isolated and identified as encoding claudin 28b, an integral component of the tight junction. Immunohistochemical reactivity to claudin 28b protein was concentrated in a circumferential ring colocalized to the cortical F-actin ring. To study the contribution of this isoform to changes in transepithelial resistance and Phenol Red diffusion under apical hypo-or hyperosmotic exposure we quantified the fluorescence signal of this claudin isoform in immunohistochemical stainings together with the fluorescence of phalloidin-probed F-actin. Upon hypo-osmotic stress claudin 28b fluorescence and epithelial tightness remained stable. Under hyperosmotic stress, the presence of claudin 28b at the junction significantly decreased, and epithelial tightness was severely reduced. Cortical F-actin fluorescence increased upon hypo-osmotic stress, whereas hyperosmotic stress led to a separation of cortical F-actin rings and the number of apical crypt-like pores increased. Addition of cortisol to the basolateral medium attenuated cortical F-actin separation and pore formation during hyperosmotic stress and reduced claudin 28b in junctions except after recovery of cells from exposure to freshwater. Our results showed that short-term salinity stress response in cultured trout gill cells was dependent on a dynamic remodeling of tight junctions, which involves claudin 28b and the supporting F-actin ring

  2. Gill's 'History' restored

    NASA Astrophysics Data System (ADS)

    Hurn, Mark

    2009-06-01

    Note about the restoration of the copy of Sir David Gill's 'A History and Description of the Royal Observatory, Cape of Good Hope' in the Library of the Institute of Astronomy, Cambridge. The book was restored with funds provided by the SHA in thanks for facilities for meetings provided to the Institute.

  3. 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

  4. Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia.

    PubMed

    Schnell, Sabine; Stott, Lucy C; Hogstrand, Christer; Wood, Chris M; Kelly, Scott P; Pärt, Peter; Owen, Stewart F; Bury, Nic R

    2016-03-01

    This protocol describes how to reconstruct and culture the freshwater rainbow trout gill epithelium on flat permeable membrane supports within cell culture inserts. The protocol describes gill cell isolation, cultured gill epithelium formation, maintenance, monitoring and preparation for use in experimental procedures. To produce a heterogeneous gill epithelium, as seen in vivo, seeding of isolated gill cells twice over a 2-d period is required. As a consequence, this is termed the double-seeded insert technique. Approximately 5-12 d after cell isolation and seeding, preparations develop electrically tight gill epithelia that can withstand freshwater on the apical cell surface. The system can be used to study freshwater gill physiology, and it is a humane alternative for toxicity testing, bioaccumulation studies and environmental water quality monitoring. PMID:26866792

  5. Cell-host, LINE and environment

    PubMed Central

    Del Re, Brunella; Giorgi, Gianfranco

    2013-01-01

    Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship. PMID:23734298

  6. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    PubMed

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment. PMID:27312328

  7. 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.

  8. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  9. Neuroepithelial oxygen chemoreceptors of the zebrafish gill

    PubMed Central

    Jonz, Michael G; Fearon, Ian M; Nurse, Colin A

    2004-01-01

    In aquatic vertebrates, hypoxia induces physiological changes that arise principally from O2 chemoreceptors of the gill. Neuroepithelial cells (NECs) of the zebrafish gill are morphologically similar to mammalian O2 chemoreceptors (e.g. carotid body), suggesting that they may play a role in initiating the hypoxia response in fish. We describe morphological changes of zebrafish gill NECs following in vivo exposure to chronic hypoxia, and characterize the cellular mechanisms of O2 sensing in isolated NECs using patch-clamp electrophysiology. Confocal immunofluorescence studies indicated that chronic hypoxia (PO2 = 35 mmHg, 60 days) induced hypertrophy, proliferation and process extension in NECs immunoreactive for serotonin or synaptic vesicle protein (SV2). Under voltage clamp, NECs responded to hypoxia (PO2 = 25–140 mmHg) with a dose-dependent decrease in K+ current. The current–voltage relationship of the O2-sensitive current (IKO2) reversed near EK and displayed open rectification. Pharmacological characterization indicated that IKO2 was resistant to 20 mm tetraethylammonium (TEA) and 5 mm 4-aminopyridine (4-AP), but was sensitive to 1 mm quinidine. In current-clamp recordings, hypoxia produced membrane depolarization associated with a conductance decrease; this depolarization was blocked by quinidine, but was insensitive to TEA and 4-AP. These biophysical and pharmacological characteristics suggest that hypoxia sensing in zebrafish gill NECs is mediated by inhibition of a background K+ conductance, which generates a receptor potential necessary for neurosecretion and activation of sensory pathways in the gill. This appears to be a fundamental mechanism of O2 sensing that arose early in vertebrate evolution, and was adopted later in mammalian O2 chemoreceptors. PMID:15331683

  10. 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.

  11. Breast cancer cell lines: friend or foe?

    PubMed Central

    Burdall, Sarah E; Hanby, Andrew M; Lansdown, Mark RJ; Speirs, Valerie

    2003-01-01

    The majority of breast cancer research is conducted using established breast cancer cell lines as in vitro models. An alternative is to use cultures established from primary breast tumours. Here, we discuss the pros and cons of using both of these models in translational breast cancer research. PMID:12631387

  12. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been widely done using labor intensive and cytotoxic liposome-based transfection reagents....

  13. 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.

  14. Effects of certain chemicals on mucus-producing cells of Petromyzon marinus

    USGS Publications Warehouse

    Sawyer, Philip J.

    1959-01-01

    Tissue samples that contained slime-secreting cells were taken from the gills and epidermis of larval lampreys that had been poisoned by several compounds. Histochemical treatment of these pathological tissues helped delineate the fate of these mucus-producing areas of the ammocetes. It was shown that the slime-secreting cells, located at the tips of the gill filaments, lining the gill chamber, and scattered throughout the epidermis reacted differently to the same toxicant. The secretory cells of the gills were, without exception, the most sensitive to chemical attack.

  15. A human gallbladder adenocarcinoma cell line.

    PubMed

    Morgan, R T; Woods, L K; Moore, G E; McGavran, L; Quinn, L A; Semple, T U

    1981-06-01

    A continuous cell line, COLO 346, was established from a liver metastasis in a patient with adenocarcinoma of the gallbladder. COLO 346 grew as an adherent monolayer of pleomorphic epithelioid cells. COLO 346 cells produced esterone, but no estradiol, progesterone, or cortisol. No adrenocorticotropic hormones, beta-subunit of human chorionic gonadotropin, carcinoembryonic antigen, or alpha-fetoprotein production by the cells was detected. Cell doubling time was 36 h. Seven allelic isozymes were assayed. COLO 346 had a chromosome mode of 74 at 21 months postestablishment with 6 marker chromosomes present in 100% of the cells analyzed. COLO 346 has been in continuous culture for over 2 yr and is available to other investigators for their studies. PMID:7262900

  16. Antiproliferative efficacy of Tabernaemontana divaricata against HEP2 cell line and Vero cell line

    PubMed Central

    Kumar, Arvind; Selvakumar, S.

    2015-01-01

    Background: Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2). Materials and Method: The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability. Results: The extract on Hep 2 cell line up to 7.8 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Conclusion: Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line). PMID:26109773

  17. Characterization of swine testicular cell line as immature porcine Sertoli cell line.

    PubMed

    Ma, Changping; Song, Huibin; Guan, Kaifeng; Zhou, Jiawei; Xia, Xuanyan; Li, Fenge

    2016-04-01

    Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions. PMID:26744029

  18. A human gallbladder adenocarcinoma cell line.

    PubMed

    Johzaki, H; Iwasaki, H; Nishida, T; Isayama, T; Kikuchi, M

    1989-12-01

    A cell strain (FU-GBC-1) was established from cancerous ascites of a 68-year-old male patient with well-differentiated adenocarcinoma of the gallbladder. By light and electron microscopy, the cultured cells showed the morphologic features of adenocarcinoma characterized by gland-like structures, intracellular microcystic spaces, and mucous production. Immunoperoxidase stains showed that FU-GBC-1 cells expressed several epithelial tumor antigens including CA 19-9, carcinoembryonic antigen (CEA), and epithelial membrane antigen (EMA). The cell strain has been in continuous culture up to passage 44 for 1 1/2 years, with the population doubling time of 120 hours. The cytogenetic analysis by a G-band technique showed a constant loss of chromosome Y in FU-GBC-1 cells. The modal chromosome number at passage 12 was 82 with a range of 77 to 85. Flow cytometry with an ethidium bromide technique additionally confirmed aneuploid DNA content (4C) in the cultured cells at passage 12 and 35. Inoculation of FU-GBC-1 cells into the dermis of BALB/c nude mice produced transplantable adenocarcinoma identical to the original tumor. Because no continuous cell lines of the well-differentiated type of gallbladder adenocarcinoma have been reported in the literature currently, the newly established cell strain we report may yield a useful system for studying the morphologic and biologic characteristics of gallbladder adenocarcinoma. PMID:2680052

  19. Effect of copper sulfate upon gill secretory cell nature, mucin production and mucin composition in Mytilus edulis with notes on Modiolus modiolus

    SciTech Connect

    Clements, J.R.

    1983-01-01

    Mussels secrete increased amounts of mucin upon exposure to copper sulfate concentrations equal to or greater than 3 ppm. The threshold level of copper necessary to elicit this response is higher if the animals are exposed to copper sulfate in filtered rather than in unfiltered seawater. At high levels of copper sulfate there is an increase in the relative hydration of the gill tissue.

  20. Microfluidic system with integrated electroosmotic pumps, concentration gradient generator and fish cell line (RTgill-W1)--towards water toxicity testing.

    PubMed

    Glawdel, Tomasz; Elbuken, Caglar; Lee, Lucy E J; Ren, Carolyn L

    2009-11-21

    This study presents a microfluidic system that incorporates electroosmotic pumps, a concentration gradient generator and a fish cell line (rainbow trout gill) to perform toxicity testing on fish cells seeded in the system. The system consists of three mechanical components: (1) a toxicity testing chip containing a microfluidic gradient generator which creates a linear concentration distribution of toxicant in a cell test chamber, (2) an electroosmotic (EO) pump chip that controls the flow rate and operation of the toxicity chip, and (3) indirect reservoirs that connect the two chips allowing for the toxicant solution to be pumped separately from the electroosmotic pump solution. The flow rate and stability of the EO pumps was measured and tested by monitoring the gradient generator using fluorescence microscopy. Furthermore, a lethality test was performed with this system setup using a rainbow trout gill cell line (RTgill-W1) as the test cells and sodium dodecyl sulfate as a model toxicant. A gradient of sodium dodecyl sulfate, from 0 to 50 microg mL(-1), was applied for 1 hr to the attached cells, and the results were quantified using a Live/Dead cell assay. This work is a preliminary study on the application of EO pumps in a living cell assay, with the potential to use the pumps in portable water quality testing devices with RTgill-W1 cells as the biosensors. PMID:19865731

  1. 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

  2. Permanently Blocked Stem Cells Derived from Breast Cancer Cell Lines

    PubMed Central

    Sajithlal, Gangadharan B.; Rothermund, Kristi; Zhang, Fang; Dabbs, David J.; Latimer, Jean J.; Grant, Stephen G.; Prochownik, Edward V.

    2016-01-01

    Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. An unanticipated result, however, was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immuno-compromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs. PMID:20506227

  3. Argyrophilic nucleolar organizer regions (AgNORs) in interphases and metaphases of normal and neoplastic gill cells of Macoma balthica (Bivalvia: Tellinidae) from the Gulf of Gdansk, Baltic Sea.

    PubMed

    Smolarz, K; Wolowicz, M; Thiriot-Quiévreux, C

    2003-10-24

    Chromosome analysis of gill cells of different populations of Macoma balthica (L.) from the Bay of Gdansk (Baltic Sea) revealed 2 clam categories, 1 with neoplastic features and 1 without. Silver-staining was performed on interphase and metaphase cells of both categories. The mean argyrophilic nucleolar organizer region (AgNOR) count per abnormal interphase cell was significantly higher than in normal interphase cells. Normal silver-stained metaphases had 3 nucleolar organizer region (NOR) chromosome phenotypes. The location of the NORs in the most frequent phenotype (55.6% in 54 metaphases scored) was interstitial on the largest metacentric chromosome pair, Pair No. 1. Abnormal silver-stained metaphases had a higher number of active NOR sites. Different phenotypes were observed (frequency greater than 10% for 67 metaphases scored); 2 were similar to those in normal metaphases and 5 were ectopic. The higher activity of AgNORs observed in abnormal cells confirmed the diagnosis of malignant neoplasia. PMID:14667039

  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. Detection algorithm for the validation of human cell lines.

    PubMed

    Eltonsy, Névine; Gabisi, Vivian; Li, Xuesong; Russe, K Blair; Mills, Gordon B; Stemke-Hale, Katherine

    2012-09-15

    Cell lines are an important tool in understanding all aspects of cancer growth, development, metastasis and tumor cell death. There has been a dramatic increase in the number of cell lines and diversity of the cancers they represent; however, misidentification and cross-contamination of cell lines can lead to erroneous conclusions. One method that has gained favor for authenticating cell lines is the use of short tandem repeats (STR) to generate a unique DNA profile. The challenge in validating cell lines is the requirement to compare the large number of existing STR profiles against cell lines of interest, particularly when considering that the profiles of many cell lines have drifted over time and original samples are not available. We report here methods that analyze the variations and the proportional changes extracted from tetra-nucleotide repeat regions in the STR analysis. This technique allows a paired match between a target cell line and a reference database of cell lines to find cell lines that match within a user designated percentage cut-off quality matrix. Our method accounts for DNA instability and can suggest whether the target cell lines are misidentified or unstable. PMID:22419365

  6. Personalized chemotherapy profiling using cancer cell lines from selectable mice

    PubMed Central

    Kamiyama, Hirohiko; Rauenzahn, Sherri; Shim, Joong Sup; Karikari, Collins A.; Feldmann, Georg; Hua, Li; Kamiyama, Mihoko; Schuler, F. William; Lin, Ming-Tseh; Beaty, Robert M.; Karanam, Balasubramanyam; Liang, Hong; Mullendore, Michael E.; Mo, Guanglan; Hidalgo, Manuel; Jaffee, Elizabeth; Hruban, Ralph H.; Jinnah, H. A.; Roden, Richard B. S.; Jimeno, Antonio; Liu, Jun O.; Maitra, Anirban; Eshleman, James R.

    2013-01-01

    Purpose High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult, because contaminating stromal cells overgrow the malignant cells. Experimental Design We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified seventy-seven FDA approved drugs with activity, including two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. PMID:23340293

  7. Gemcitabine induces cell senescence in human pancreatic cancer cell lines.

    PubMed

    Song, Yao; Baba, Tomohisa; Mukaida, Naofumi

    2016-08-26

    Patients with pancreatic ductal adenocarcinoma (PDAC) commonly require chemotherapy because they frequently develop metastatic disease or locally advanced tumors. Gemcitabine, an analogue of cytosine arabinoside, is commonly used for PDAC treatment. We observed that gemcitabine induced senescence phenotypes characterized by enhanced senescence-associated β-galactosidase (SA β-Gal) staining and increased expression of senescence-associated molecules in two human pancreatic cancer cell lines, Miapaca-2 and Panc-1, which exhibit resistance to gemcitabine but not L3.pl cells with a high sensitivity to gemcitabine. Gemcitabine-induced cell senescence can be inhibited by reactive oxygen species inhibitor, N-acetyl cysteine. Although gemcitabine also enhanced CXCL8 expression, anti-CXCL8 antibody failed to reduce gemcitabine-induced increases in SA β-Gal-positive cell numbers. These observations would indicate that cell senescence can proceed independently of CXCL8 expression, a characteristic feature of senescence-associated secretion phenotype. PMID:27311854

  8. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  9. 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. PMID:24297409

  10. ULTRASTRUCTURAL STUDY OF LESIONS IN GILLS OF A MARINE SHRIMP EXPOSED TO CADMIUM

    EPA Science Inventory

    Pathologic black gills of pink shrimp, Penaeus duorarum, exposed to 763 micrograms/l of cadmium chloride for 15 days were studied with transmission electron microscopy and were compared with normal gills of control pink shrimp. Local as well as extensive areas of cell death and n...

  11. 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

  12. Cell line banks and their role in cancer research.

    PubMed

    Hay, R J; Reid, Y A; McClintock, P R; Chen, T R; Macy, M L

    1996-01-01

    The utility of centralized cell banks in providing reference cultures for cancer research is reviewed. Procedures applied at The American Type Culture Collection in development, maintenance and expansion of such a resource are discussed for example, with emphasis on human tumor cell lines. The various categories of cell-line holdings are explained, and status with regard both to the numbers of lines available and distribution experienced are documented. The locations of other national cell repositories plus contact data are provided. PMID:8806094

  13. Characterization of abalone Haliotis tuberculata-Vibrio harveyi interactions in gill primary cultures.

    PubMed

    Pichon, Delphine; Cudennec, Benoit; Huchette, Sylvain; Djediat, Chakib; Renault, Tristan; Paillard, Christine; Auzoux-Bordenave, Stéphanie

    2013-10-01

    The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell-V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments. PMID:23756730

  14. A western type of bacterial gill disease

    USGS Publications Warehouse

    Fish, F.F.

    1935-01-01

    The first reference to a pathological condition of the gill tissues of salmonid fishes was made by Osburn in 1910. This author in describing a progressive infolding of the opercula of trout, commonly known to hatcherymen as "short gill covers," mentioned a marked proliferation on the gill epithelium as accompanying this condition. Osburn assumed that the club-like appearance of the gill filaments due to the proliferated epithelium was the result of continual irritation of the delicate gill tissue in the absence of the usual protection offered by the normal opercula. Although such a conclusion seems quite logical, it is also possible that Osburn was dealing with "short gill covers" complicated by the unknown bacterial gill disease which was subsequently described by Davis.

  15. Development and characterization of a new human hepatic cell line.

    PubMed

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  16. Development and characterization of a new human hepatic cell line

    PubMed Central

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  17. Differential signaling of the GnRH receptor in pituitary gonadotrope cell lines and prostate cancer cell lines

    PubMed Central

    Sviridonov, Ludmila; Dobkin-Bekman, Masha; Shterntal, Boris; Przedecki, Fiorenza; Formishell, Linor; Kravchook, Shani; Navi, Liat Rahamim-Ben; Bar-Lev, Tali Hana; Kazanietz, Marcelo G.; Yao, Zhong; Seger, Rony; Naor, Zvi

    2014-01-01

    The GnRH receptor (GnRHR) mediates the pituitary functions of GnRH, as well as its anti-proliferative effects in sex hormone-dependent cancer cells. Here we compare the signaling of GnRHR in pituitary gonadotrope cell lines vs. prostate cancer cell lines. We first noticed that the expression level of PKCα, PKCβII and PKCε is much higher in αT3-1 and LβT2 gonadotrope cell lines vs. LNCaP and DU-145 cell lines, while the opposite is seen for PKCδ. Activation of PKCα, PKCβII and PKCε by GnRH is relatively transient in αT3-1 and LβT2 gonadotrope cell lines and more prolonged in LNCaP and DU-145 cell lines. On the otherhand, the activation and re-distribution of the above PKCs by PMA was similar for both gonadotrope cell lines and prostate cancer cell lines. Activation of ERK1/2 by GnRH and PMA was robust in the gonadotrope cell lines, with a smaller effect observed in the prostate cancer cell lines. The Ca2+ ionophore A23187 stimulated ERK1/2 in gonadotrope cell lines but not in prostate cancer cell lines. GnRH, PMA and A23187 stimulated JNK activity in gonadotrope cell lines, with a more sustained effect in prostate cancer cell lines. Sustained activation of p38 was observed for PMA and A23187 in Du-145 cells, while p38 activation by GnRH, PMA and A23187 in LβT2 cells was transient. Thus, differential expression and re-distribution of PKCs by GnRH and the transient vs. the more sustained nature of the activation of the PKC-MAPK cascade by GnRH in gonadotrope cell lines vs. prostate cancer cell lines respectively, may provide the mechanistic basis for the cell context-dependent differential biological responses observed in GnRH interaction with pituitary gonadotropes vs. prostate cancer cells. PMID:23380421

  18. Gill (Na+ +K+)-ATPase involvement and regulation during salmonid adaptation to salt water.

    PubMed

    Borgatti, A R; Pagliarani, A; Ventrella, V

    1992-08-01

    1. The involvement of gill (Na+ +K+)-ATPase in salmonid adaptation to salt water (SW) is discussed. 2. Gill (Na+ +K+)-ATPase increase during SW adaptation is mainly related to the increased number and complexity of chloride cells deputed to salt extrusion. 3. The temporal relationships between serum peaks of thyroid hormones, cortisol, growth hormone, prolactin and gill (Na+ +K+)-ATPase rise during salmonid smoltification, suggest a hormonal involvement in the enzyme stimulation and thus in the acquirement of SW tolerance. 4. Literature on gill (Na+ +K+)-ATPase response to hormonal treatment is reviewed. The effects produced on gill (Na+ +K+)-ATPase and chloride cells by exogenous hormones point out a complex inter-relationship between the hormones considered. The mechanisms involved in hormonal regulation of the enzyme remain a matter of debate. PMID:1355028

  19. Development of oxygen sensing in the gills of zebrafish.

    PubMed

    Jonz, Michael G; Nurse, Colin A

    2005-04-01

    Previous studies have described the morphology, innervation and O(2)-chemoreceptive properties of neuroepithelial cells (NECs) of the zebrafish gill filaments. The present work describes the ontogenesis of these cells, and the formation of functional O(2)-sensing pathways in developing zebrafish. Confocal immunofluorescence was performed on whole-mount gill preparations using antibodies against serotonin (5-HT) and a zebrafish-derived neuronal marker (zn-12) to identify the appearance and innervation of gill NECs during larval stages. NECs were first expressed in gill filament primordia of larvae at 5 days postfertilization (d.p.f.) and were fully innervated by 7 d.p.f. In vivo ventilation frequency analysis revealed that a behavioural response to hypoxia (11.2+/-2.8 min(-1)) developed in embryos as early as 2 d.p.f., and a significant increase (P<0.05) in the ventilatory response to hypoxia (200.8+/-23.0 min(-1)) coincided with innervation of NECs of the filaments. In addition, exogenous application of quinidine, a blocker of O(2)-sensitive background K(+) channels in NECs, induced hyperventilation in adults in a dose-dependent manner and revealed the development of a quinidine-sensitive ventilatory response in 7 d.p.f. larvae. This study shows that NEC innervation in the gill filaments may account for the development of a functional O(2)-sensing pathway and the hyperventilatory response to hypoxia in zebrafish larvae. At earlier stages, however, O(2)-sensing must occur through another pathway. The possibility that a new type of 5-HT-positive NEC of the gill arches may account for this earlier hypoxic response is discussed. PMID:15802677

  20. Apoptosis and necroptosis are induced in rainbow trout cell lines exposed to cadmium.

    PubMed

    Krumschnabel, Gerhard; Ebner, Hannes L; Hess, Michael W; Villunger, Andreas

    2010-08-01

    Cadmium is an important environmental toxicant that can kill cells. A number of studies have implicated apoptosis as well as necrosis and, most recently, a form of programmed necrosis termed necroptosis in the process of cadmium-mediated toxicity, but the exact mechanism remains ill-defined and may depend on the affected cell type. This study investigated which mode of cell death may be responsible for cell death induction in cadmium-exposed trout cell lines from gill and liver and if this cell death was sensitive to inhibitors of necroptosis or apoptosis, respectively. It was observed that intermediate levels of cadmium that killed approximately 50% of the cells over 96-120h of exposure caused cell death that morphologically resembled apoptosis and was associated with an increase of apoptotic markers such as the number of cells with diminished DNA content (sub-G1 cells), condensed or fragmented nuclei, and elevation of caspase-3 activity. At the same time, however, cells also lost plasma membrane integrity, as indicated by uptake of propidium iodide, showed a decrease of ATP levels and mitochondrial membrane potential, and displayed cell swelling, signs associated with secondary necrosis, or equally possible, necroptotic cell death. Importantly, many of these alterations were at least partly inhibited by the necroptosis inhibitor necrostatin-1 and were to a lesser extent also sensitive to the pan-caspase inhibitor zVAD-fmk, indicating that multiple modes of cell death are concurrently induced in cadmium-exposed trout cells, including necroptosis and apoptosis. Cell death appeared to lack concurrent radical formation, consistent with genetically regulated necroptotic cell death, but was characterized by the rapid induction of DNA damage markers, and the early onset of disintegration of the Golgi complex. Comparative experiments evaluating copper-toxicity indicated that in comparison to cadmium much higher concentrations of this metal were required to induce cell

  1. Analysis of three marine fish cell lines by rapd assay.

    PubMed

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  2. Establishment and characterization of unique human gallbladder cancer cell lines.

    PubMed

    Ghosh, Mila; Koike, Naoto; Yanagimoto, Go; Tsunoda, Shin-Ichi; Kaul, Sunil; Hirano, Takashi; Emura, Fabian; Kashiwagi, Hironobu; Kawamoto, Toru; Ohkohchi, Nobuhiro; Saijo, Kaoru; Ohno, Tadao; Miwa, Masanao; Todoroki, Takeshi

    2004-05-01

    Gallbladder cancer has a dismal prognosis. Understanding the disease at the biological, genetic, molecular, cellular, and clinical level is essential for effective diagnostics and therapeutics. However, the currently established gallbladder cell lines are insufficient for better understanding and further research. The aim of our present study was to establish and characterize human gallbladder cancer cell lines. We established 5 cell lines from resected specimens of gallbladder cancers. These cell lines revealed typical tumor histopathological characteristics. We examined growth characteristics and the colony-forming ability of established cell lines in terms of their cell cycle parameters, expression of tumor markers (carcinoembryonic antigen; CEA, carbohydrated antigen 19-9; CA19-9, MUC-1 and c-kit) and the oncogene c-erbB2 by flow cytometer. Comparative genomic hybridization (CGH) analysis with specific gene probes was performed to detect changes in the gene copy numbers. Human origin of cell lines was confirmed by chromosomal analysis. Cells maintained differentiation characteristics of the original tumors. The doubling time of different cell lines varied from 30 to 96 h. All 5 cell lines formed colonies in the colony forming assays and expressed CEA, CA19-9, MUC-1 and the oncogene c-erbB2 and showed chromosomal aneuploidy. CGH analysis demonstrated gain of chromosomal region bearing SRC, RAB1, and PAP in all cell lines and hTERT in 4 cell lines. These newly established cell lines might serve as a useful model for studying the molecular pathogenesis of gallbladder cancer. Furthermore, they may serve as a model for testing new therapeutics against gallbladder cancer. These chromosomal aberrations and imbalances provide a starting point for molecular analyses of genomic regions and genes in gallbladder carcinogenesis. PMID:15067341

  3. 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.

  4. 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.

  5. The pursuit of ES cell lines of domesticated ungulates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features.

    PubMed

    Dangles-Marie, Virginie; Pocard, Marc; Richon, Sophie; Weiswald, Louis-Bastien; Assayag, Franck; Saulnier, Patrick; Judde, Jean-Gabriel; Janneau, Jean-Louis; Auger, Nathalie; Validire, Pierre; Dutrillaux, Bernard; Praz, Françoise; Bellet, Dominique; Poupon, Marie-France

    2007-01-01

    Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease. PMID:17210723

  7. High incidence of TERT mutation in brain tumor cell lines.

    PubMed

    Johanns, Tanner M; Fu, Yujie; Kobayashi, Dale K; Mei, Yu; Dunn, Ian F; Mao, Diane D; Kim, Albert H; Dunn, Gavin P

    2016-07-01

    TERT promoter gene mutations are highly recurrent in malignant glioma. However, little information exists regarding their presence in experimental brain tumor models. To better characterize systems in which TERT mutation studies could be appropriately modeled experimentally, the TERT promoter was examined by conventional sequencing in primary brain tumor initiating cells (BTIC), two matched recurrent BTIC lines, a panel of established malignant glioma cell lines, and two meningioma cell lines. Telomerase gene expression was examined by quantitative PCR. We found that all glioblastoma BTIC lines harbored a TERT mutation, which was retained in two patient-matched recurrent BTIC. The TERT C228T or C250T mutation was found in 33/35 (94 %) of established malignant glioma cell lines and both meningioma cell lines examined. Brain tumor cell lines expressed variably high telomerase levels. Thus, a high percentage of glioma cell lines, as well as two meningioma cell lines, harbors TERT mutations. These data characterize tractable, accessible models with which to further explore telomerase biology in these tumor types. PMID:26960334

  8. Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia.

    PubMed

    Bui, Phuong; Bagherie-Lachidan, Mazdak; Kelly, Scott P

    2010-04-12

    A primary cultured gill epithelium from the puffer fish Tetraodon nigroviridis was developed to examine the corticosteroid regulation of claudin isoform mRNA abundance in fish gills. Preparations were composed of polygonal epithelial cells exhibiting concentric apical microridges and zonula occludens-1 immunoreactivity along cell margins. No evidence was found to indicate the presence of Na(+)-K(+)-ATPase-immunoreactive or mitochondria-rich cells in cultured preparations. Therefore, epithelia appear to be composed of gill pavement cells (PVCs) only. An RT-PCR profile of 12 salinity responsive gill claudin tight junction (TJ) proteins (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b) revealed the absence of Tncldn6, -10d and -10e in cultured epithelia, suggesting that these isoforms are not associated with gill PVCs. Cortisol treatment of cultured epithelia dose-dependently increased or decreased mRNA abundance of select claudin isoforms. Transcript abundance of several claudin isoforms was unaffected by cortisol treatment. These data provide evidence for the cell specific distribution of claudins in fish gills and suggest that heterogeneous alterations in the abundance of select claudin isoforms contribute to the corticosteroid regulation of gill permeability. PMID:19969041

  9. Authentication of the R06E Fruit Bat Cell Line

    PubMed Central

    Jordan, Ingo; Munster, Vincent J.; Sandig, Volker

    2012-01-01

    Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery. PMID:22754654

  10. Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

    PubMed Central

    Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

    2013-01-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

  11. A shared role for sonic hedgehog signalling in patterning chondrichthyan gill arch appendages and tetrapod limbs.

    PubMed

    Gillis, J Andrew; Hall, Brian K

    2016-04-15

    Chondrichthyans (sharks, skates, rays and holocephalans) possess paired appendages that project laterally from their gill arches, known as branchial rays. This led Carl Gegenbaur to propose that paired fins (and hence tetrapod limbs) originally evolved via transformation of gill arches. Tetrapod limbs are patterned by asonic hedgehog(Shh)-expressing signalling centre known as the zone of polarising activity, which establishes the anteroposterior axis of the limb bud and maintains proliferative expansion of limb endoskeletal progenitors. Here, we use loss-of-function, label-retention and fate-mapping approaches in the little skate to demonstrate that Shh secretion from a signalling centre in the developing gill arches establishes gill arch anteroposterior polarity and maintains the proliferative expansion of branchial ray endoskeletal progenitor cells. These findings highlight striking parallels in the axial patterning mechanisms employed by chondrichthyan branchial rays and paired fins/limbs, and provide mechanistic insight into the anatomical foundation of Gegenbaur's gill arch hypothesis. PMID:27095494

  12. AB241. Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P

    PubMed Central

    Huang, Bin; Wang, Dao-Hu; Chen, Jun-Xing; Qiu, Shao-Peng

    2016-01-01

    Background Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Methods We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Results Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells among five cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Conclusions Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

  13. Permissiveness of human hepatoma cell lines for HCV infection

    PubMed Central

    2012-01-01

    Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). Results We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection. PMID:22273112

  14. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  15. Derivation of the human embryonic stem cell line RCM1.

    PubMed

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  16. 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.

  17. 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.

  18. Optimal lamellar arrangement in fish gills

    PubMed Central

    Park, Keunhwan; Kim, Wonjung; Kim, Ho-Young

    2014-01-01

    Fish respire through gills, which have evolved to extract aqueous oxygen. Fish gills consist of filaments with well-ordered lamellar structures, which play a role in maximizing oxygen diffusion. It is interesting that when we anatomically observe the gills of various fish species, gill interlamellar distances (d) vary little among them, despite large variations in body mass (Mb). Noting that the small channels formed by densely packed lamellae cause significant viscous resistance to water flow, we construct and test a model of oxygen transfer rate as a function of the lamellar dimensions and pumping pressure, which allows us to predict the optimal interlamellar distance that maximizes the oxygen transfer rate in the gill. Comparing our theory with biological data supports the hypothesis that fish gills have evolved to form the optimal interlamellar distances for maximizing oxygen transfer. This explains the weak scaling dependence of d on Mb: d ∼ Mb1/6. PMID:24847065

  19. The effects of oncolytic reovirus in canine lymphoma cell lines.

    PubMed

    Hwang, C C; Umeki, S; Igase, M; Coffey, M; Noguchi, S; Okuda, M; Mizuno, T

    2016-08-01

    Reovirus is a potent oncolytic virus in many human neoplasms that has reached phase II and III clinical trials. Our laboratory has previously reported the oncolytic effects of reovirus in canine mast cell tumour (MCT). In order to further explore the potential of reovirus in veterinary oncology, we tested the susceptibility of reovirus in 10 canine lymphoma cell lines. Reovirus-induced cell death, virus replication and infectivity were confirmed in four cell lines with variable levels of susceptibility. The level of Ras activation varied among the cell lines with no correlation with reovirus susceptibility. Reovirus-susceptible cell lines underwent apoptosis as proven by propidium iodide (PI) staining, Annexin V-FITC/PI assay, cleavage of PARP and inhibition of cell death by caspase inhibitor. A single intratumoral injection of reovirus suppressed the growth of canine lymphoma subcutaneous tumour in NOD/SCID mice. Unlike canine MCT, canine lymphoma is less susceptible to reovirus. PMID:25319493

  20. CACO-2 CELL LINES IN DRUG DISCOVERY- AN UPDATED PERSPECTIVE

    PubMed Central

    Kumar, Kalyan K.V; Karnati, Swathi; Reddy, Mamatha B; Chandramouli, R

    2010-01-01

    Cell lines are the invitro models used for the drug permeability studies in the preclinical and clinical phases of the drug discovery. Cell line models are simple and quick to use and avoids the usage of animal models for pharmacological and toxicological studies and hence cost effective, produce reliable and reproducible results for understanding and evaluating the permeability characteristics of the potential lead drug candidates. Different cell line models used in the drug permeability studies, their characteristics has been summarized emphasizing on CACO-2. By virtue of its merits, CACO-2 cell line development, transport experiments, automated assays, optimization of experimental conditions and mechanistic uses of CACO-2 cell lines dealt comprehensively in the following context. PMID:24825967

  1. Derivation of Genea057 human embryonic stem cell line.

    PubMed

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

    2016-01-01

    The Genea057 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea057 was demonstrated with 97% of cells expressing Nanog, 81% Oct4, 75% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.59 and Novelty score of 1.32. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345782

  2. Derivation of Genea042 human embryonic stem cell line.

    PubMed

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

    2016-03-01

    The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345994

  3. Derivation of Genea002 human embryonic stem cell line.

    PubMed

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

    2016-01-01

    The Genea002 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype by CGH and male Allele pattern through STR analysis. Pluripotency of Genea002 was demonstrated with 75% of cells expressing Nanog, 93% Oct4, 83% Tra1-60 and 98% SSEA4, a Pluritest pluripotency score of 24.55, Novelty score of 1.39, teratomas with tissues from all embryonic germ layers and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345802

  4. Derivation of Genea052 human embryonic stem cell line.

    PubMed

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

    2016-03-01

    The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345996

  5. Derivation of human embryonic stem cell line Genea023.

    PubMed

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

    2016-03-01

    The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346015

  6. Derivation of Genea015 human embryonic stem cell line.

    PubMed

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

    2016-03-01

    The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346028

  7. Derivation of human embryonic stem cell line Genea022.

    PubMed

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

    2016-03-01

    The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346017

  8. Derivation of Genea047 human embryonic stem cell line.

    PubMed

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

    2016-03-01

    The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345995

  9. Derivation of Genea043 human embryonic stem cell line.

    PubMed

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

    2016-01-01

    The Genea043 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea043 was demonstrated with 92% of cells expressing Nanog, 95% Oct4, 61% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 31.74, Novelty score of 1.2 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345801

  10. Derivation of Genea016 human embryonic stem cell line.

    PubMed

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

    2016-01-01

    The Genea016 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea016 was demonstrated with 77% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 28.4, Novelty score of 1.37 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345780

  11. Regulatory networks define phenotypic classes of human stem cell lines

    PubMed Central

    Müller, Franz-Josef; Laurent, Louise C.; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Schwartz, Philip H.; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine1, 2 have highlighted the need for a general, reproducible method for classification of these cells3. We report here the creation and analysis of a database of global gene expression profiles (“Stem Cell Matrix”) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent, and differentiated cell types. Using an unsupervised clustering method4, 5 to categorize a collection of ~150 cell samples, we discovered that pluripotent stem cell lines group together, while other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis6 we uncovered a protein-protein network (“PluriNet”) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas, and induced pluripotent cells). Analysis of published data showed that the PluriNet appears to be a common characteristic of pluripotent cells, including mouse ES and iPS cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotence and self-renewal are under tight control by specific molecular networks. PMID:18724358

  12. Comparison of in vitro and in vivo acute toxicity assays in Etroplus suratensis (Bloch, 1790) and its three cell lines in relation to tannery effluent.

    PubMed

    Taju, G; Majeed, S Abdul; Nambi, K S N; Sarath Babu, V; Vimal, S; Kamatchiammal, S; Hameed, A S Sahul

    2012-03-01

    Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC(50) values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r(2)=0.95-0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC(50) and the in vivo LC(50) data, were highly significant p<0.001 with r(2)=0.977, 0.968 and 0.906 for AB(50), NR(50), and CB(50), respectively. PMID:22205045

  13. 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. PMID:27263415

  14. 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. PMID:27322762

  15. Novel human bronchial epithelial cell lines for cystic fibrosis research

    PubMed Central

    Fulcher, M. L.; Gabriel, S. E.; Olsen, J. C.; Tatreau, J. R.; Gentzsch, M.; Livanos, E.; Saavedra, M. T.; Salmon, P.; Randell, S. H.

    2009-01-01

    Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ΔF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Ω·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1β, TNF-α, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development. PMID:18978040

  16. Generation and characterization of human insulin-releasing cell lines

    PubMed Central

    Labriola, Leticia; Peters, Maria G; Krogh, Karin; Stigliano, Iván; Terra, Letícia F; Buchanan, Cecilia; Machado, Marcel CC; Joffé, Elisa Bal de Kier; Puricelli, Lydia; Sogayar, Mari C

    2009-01-01

    Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction. PMID:19545371

  17. 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

  18. 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

  19. Vaccine production: upstream processing with adherent or suspension cell lines.

    PubMed

    Genzel, Yvonne; Rödig, Jana; Rapp, Erdmann; Reichl, Udo

    2014-01-01

    The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered. PMID:24297427

  20. 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. PMID:25229492

  1. Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

    PubMed

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

    2001-01-01

    33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity. PMID:11497276

  2. Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    PubMed Central

    Fausther, Michel; Goree, Jessica R.; Lavoie, Élise G.; Graham, Alicia L.; Sévigny, Jean; Dranoff, Jonathan A.

    2015-01-01

    The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5’-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis. PMID:25822334

  3. Human papillomavirus in vulvar and vaginal carcinoma cell lines.

    PubMed Central

    Hietanen, S.; Grénman, S.; Syrjänen, K.; Lappalainen, K.; Kauppinen, J.; Carey, T.; Syrjänen, S.

    1995-01-01

    A number of reports associate human papillomavirus (HPV) with cervical cancer and cancer cell lines derived from this tumour type. Considerably fewer reports have focused on the role of HPV in carcinomas from other sites of female anogenital squamous epithelia. In this study we have tested for the presence of HPV in eight low-passage vulvar carcinoma cell lines and one extensively passaged cell line, A431. One cell line from a primary vaginal carcinoma was included. The presence of the HPV was evaluated by the polymerase chain reaction (PCR), by Southern blot analysis and by two-dimensional gel electrophoresis. General primer-mediated PCR was applied by using primers from the L1 region, E1 region and HPV 16 E7 region. Southern blot hybridisation was performed under low-stringency conditions (Tm = -35 degrees C) using a whole genomic HPV 6/16/18 probe mixture and under high stringency conditions (Tm = -18 degrees C) with the whole genomic probes of HPV 16 and 33. HPV 16 E6-E7 mRNA was assessed by ribonuclease protection assay (RPA). HPV was found in only one vulvar carcinoma cell line, UM-SCV-6. The identified type, HPV 16, was integrated in the cell genome and could be amplified with all primers used. Also E6-E7 transcripts were found in these cells. Five original tumour biopsies were available from the HPV-negative cell lines for in situ hybridisation. All these were HPV negative with both the HPV 6/16/18 screening probe mixture under low stringency and the HPV 16 probe under high stringency. The results indicate that vulvar carcinoma cell lines contain HPV less frequently than cervical carcinoma cell lines and suggest that a significant proportion of vulvar carcinomas may evolve by an HPV-independent mechanism. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7599042

  4. Ultrastructural effects on gill tissues induced in red tilapia Oreochromis sp. by a waterborne lead exposure.

    PubMed

    Aldoghachi, Mohammed A; Azirun, Mohd Sofian; Yusoff, Ismail; Ashraf, Muhammad Aqeel

    2016-09-01

    Experiments on hybrid red tilapia Oreochromis sp. were conducted to assess histopathological effects induced in gill tissues of 96 h exposure to waterborne lead (5.5 mg/L). These tissues were investigated by light and scanning electron microscopy. Results showed that structural design of gill tissues was noticeably disrupted. Major symptoms were changes of epithelial cells, fusion in adjacent secondary lamellae, hypertrophy and hyperplasia of chloride cells and coagulate necrosis in pavement cells with disappearance of its microridges. Electron microscopic X-ray microanalysis of fish gills exposed to sublethal lead revealed that lead accumulated on the surface of the gill lamella. This study confirmed that lead exposure incited a difference of histological impairment in fish, supporting environmental watch over aquatic systems when polluted by lead. PMID:27579014

  5. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  6. [The effects of actovegin on cell proliferation of permanent lines].

    PubMed

    Gulevskiĭ, A K; Trifonova, A V; Lavrik, A A

    2008-01-01

    The influence of Actovegin on proliferation activity and mitotic regimen of cells of permanent lines PK-15-IEKVM and BHK-21 clone 13/04 was investigated. Addition of Actovegin into growth media containing bovine serums of different components and concentrations stimulates cell proliferation. Conclusion has been made that Actovegin can be used in cell culture biotechnology. PMID:18411759

  7. CHARACTERIZATION OF A SPONTANEOUSLY TRANSFORMED CHICKEN MONONUCLEAR CELL LINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 C or 41 C...

  8. 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

  9. An interim report on gill disease

    USGS Publications Warehouse

    Rucker, R.R.; Johnson, H.E.; Kaydas, G.M.

    1952-01-01

    GILL DISEASE among fish, a disease which is characterized by a proliferation of the gill epithelium, has been attributed to a number of different causes. Generally, there are two recognized types: the eastern or bacterial type, in which long filamentous bacteria can always be demonstrated; and the western type, in which, by definition, bacteria cannot be demonstrated.

  10. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    PubMed

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  11. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease

    PubMed Central

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-01-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  12. Global Conservation of Protein Status between Cell Lines and Xenografts.

    PubMed

    Biau, Julian; Chautard, Emmanuel; Court, Frank; Pereira, Bruno; Verrelle, Pierre; Devun, Flavien; De Koning, Leanne; Dutreix, Marie

    2016-08-01

    Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery. PMID:27567954

  13. Development of cystic fibrosis and noncystic fibrosis airway cell lines.

    PubMed

    Zabner, Joseph; Karp, Phil; Seiler, Michael; Phillips, Stacia L; Mitchell, Calista J; Saavedra, Mimi; Welsh, Michael; Klingelhutz, Aloysius J

    2003-05-01

    In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-kappaB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-kappaB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity. PMID:12676769

  14. 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. PMID:20623584

  15. Surface charge characteristics of cells from malignant cell lines and normal cell lines of the human hematopoietic system.

    PubMed

    Marikovsky, Y; Ben-Bassat, H; Leibovich, S J; Cividalli, L; Fischler, H; Danon, D

    1979-02-01

    Cells from malignant and normal lines of human hematopoietic origin were studied for their surface charge characteristics with the use of the following criteria: 1) the electron microscopic appearance of cell membranes after labeling with cationized ferritin (CF) either before or after glutaraldehyde fixation, 2) electrophoretic mobility, 3) total sialic acid content, and 4) agglutinability with poly-L-lysine (PLL). CF induced a time-dependent redistribution of surface receptors in unfixed malignant cells but not in unfixed normal cells. After 10 seconds of labeling with CF, both normal and malignant unfixed cells showed a uniform and even labeling pattern. After 5 minutes of labeling, malignant cells exhibited a highly pronounced pattern of clusters and patches, as distinct from a random and even pattern exhibited by normal cells. Both normal and malignant cells after fixation exhibited an equivalent random and even labeling pattern with CF, independent of the duration of labeling. The malignant cells studied possessed less sialic acid, had a lower electric mobility, and were agglutinated more readily with PLL than were the normal cells. PMID:310907

  16. 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.

  17. 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.

  18. DNA Fingerprinting of the NCI-60 Cell Line Panel

    PubMed Central

    Lorenzi, Philip L.; Reinhold, William C.; Varma, Sudhir; Hutchinson, Amy A.; Pommier, Yves; Chanock, Stephen J.; Weinstein, John N.

    2009-01-01

    The National Cancer Institute’s NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Since many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also demonstrate that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes. PMID:19372543

  19. DNA fingerprinting of the NCI-60 cell line panel.

    PubMed

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes. PMID:19372543

  20. Cold storage and cryopreservation of tick cell lines

    PubMed Central

    2010-01-01

    Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B.) microplus, R. (B.) decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B.) decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B.) microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B.) decoloratus, R. (B.) microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance. PMID:20388200

  1. Pharmacogenomic agreement between two cancer cell line data sets.

    PubMed

    2015-12-01

    Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics resources: The Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer databases. PMID:26570998

  2. Oxidative Stress and Nano-Toxicity Induced by TiO2 and ZnO on WAG Cell Line

    PubMed Central

    Dubey, Akhilesh; Goswami, Mukunda; Yadav, Kamalendra; Chaudhary, Dharmendra

    2015-01-01

    Metallic nanoparticles are widely used in cosmetics, food products and textile industry. These particles are known to cause respiratory toxicity and epithelial inflammation. They are eventually released to aquatic environment necessitating toxicity studies in cells from respiratory organs of aquatic organisms. Hence, we have developed and characterized a new cell line, WAG, from gill tissue of Wallago attu for toxicity assessment of TiO2 and ZnO nanoparticles. The efficacy of the cell line as an in vitro system for nanoparticles toxicity studies was established using electron microscopy, cytotoxicity assays, genotoxicity assays and oxidative stress biomarkers. Results obtained with MTT assay, neutral red uptake assay and lactate dehydrogenase assay showed acute toxicity to WAG cells with IC50 values of 25.29±0.12, 34.99±0.09 and 35.06±0.09 mg/l for TiO2 and 5.716±0.1, 3.160±0.1 and 5.57±0.12 mg/l for ZnO treatment respectively. The physicochemical properties and size distribution of nanoparticles were characterized using electron microscopy with integrated energy dispersive X-ray spectroscopy and Zetasizer. Dose dependent increase in DNA damage, lipid peroxidation and protein carbonylation along with a significant decrease in activity of Superoxide Dismutase, Catalase, total Glutathione levels and total antioxidant capacity with increasing concentration of exposed nanoparticles indicated that the cells were under oxidative stress. The study established WAG cell line as an in vitro system to study toxicity mechanisms of nanoparticles on aquatic organisms. PMID:26011447

  3. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

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

    PubMed

    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    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

  5. 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

  6. Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

    PubMed Central

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

    2013-01-01

    Abstract 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. PMID:23777253

  7. Genotypes and immunophenotypes of Hodgkin's disease-derived cell lines.

    PubMed

    Drexler, H G; Leber, B F; Norton, J; Yaxley, J; Tatsumi, E; Hoffbrand, A V; Minowada, J

    1988-06-01

    This report describes the geno- and immunophenotypic analysis of the Hodgkin's disease-derived cell lines HDLM-2, KM-H2, and L-428. The lines were all positive for the antigens CD15 (Leu-M1), CD30 (Ki-1), Hefi-1 (antigen detected by a monoclonal antibody produced against L-428), HLA class I and II, and activation/proliferation markers. The cells from all 3 cell lines lacked almost all cell lineage-associated/specific markers: HDLM-2 was only CD2+, KM-H2 was only CD9+ and CD21+, and L-428 was negative for all the specific markers tested. Genomic analysis of HDLM-2 cells revealed monoclonal rearrangements of T cell receptor beta and gamma loci and germ line configuration of immunoglobulin genes. Immunoglobulin heavy chain genes were rearranged in KM-H2 and L-428. These data suggest a possible lymphoid origin for HDLM-2, KM-H2, and L-428. Although the data presented do not provide formal proof of a lymphoid nature of Hodgkin and Reed-Sternberg cells and do not unequivocally exclude a derivation from other hematopoietic cells, extrapolation of the results from the in vitro cultures to the in vivo situation suggests a lymphoid (T or B cell) origin of these cells. PMID:3131596

  8. 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

  9. Xenotropic retrovirus Bxv1 in human pancreatic β cell lines

    PubMed Central

    Kirkegaard, Jeannette S.; Ingvarsen, Signe; Diedisheim, Marc; Bricout-Neveu, Emilie; Grønborg, Mads; Frogne, Thomas; Scharfmann, Raphael; Madsen, Ole D.; Rescan, Claude; Albagli, Olivier

    2016-01-01

    It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic β cell lines (EndoC-βH1 and EndoC-βH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-βH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-βH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent β cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and βTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-βH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-βH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices. PMID:26901817

  10. 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

  11. Why mushrooms form gills: efficiency of the lamellate morphology

    PubMed Central

    FISCHER, Mark W. F.; MONEY, Nicholas P.

    2009-01-01

    Gilled mushrooms are produced by multiple orders within the Agaricomycetes. Some species form a single array of unbranched radial gills beneath their caps, many others produce multiple files of lamellulae between the primary gills, and branched gills are also common. In this largely theoretical study we modeled the effects of different gill arrangements on the total surface area for spore production. Relative to spore production over a flat surface, gills achieve a maximum 20-fold increase in surface area. The branching of gills produces the same increase in surface area as the formation of freestanding lamellulae (short gills). The addition of lamellulae between every second gill would offer a slightly greater increase in surface area in comparison to the addition of lamellulae between every pair of opposing gills, but this morphology does not appear in nature. Analysis of photographs of mushrooms demonstrates an excellent match between natural gill arrangements and configurations predicted by our model. PMID:20965062

  12. Why mushrooms form gills: efficiency of the lamellate morphology.

    PubMed

    Fischer, Mark W F; Money, Nicholas P

    2010-01-01

    Gilled mushrooms are produced by multiple orders within the Agaricomycetes. Some species form a single array of unbranched radial gills beneath their caps, many others produce multiple files of lamellulae between the primary gills, and branched gills are also common. In this largely theoretical study we modeled the effects of different gill arrangements on the total surface area for spore production. Relative to spore production over a flat surface, gills achieve a maximum 20-fold increase in surface area. The branching of gills produces the same increase in surface area as the formation of free-standing lamellulae (short gills). The addition of lamellulae between every second gill would offer a slightly greater increase in surface area in comparison to the addition of lamellulae between every pair of opposing gills, but this morphology does not appear in nature. Analysis of photographs of mushrooms demonstrates an excellent match between natural gill arrangements and configurations predicted by our model. PMID:20965062

  13. 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

  14. Effect of dehydrodidemnin B on human colon carcinoma cell lines.

    PubMed

    Lobo, C; García-Pozo, S G; Núñez de Castro, I; Alonso, F J

    1997-01-01

    Didemnins are cytotoxic agents belonging to a depsipeptide family isolated from marine tunicates. In the present study, a new member, dehydrodidemnin B (DDB), isolated from the mediterranean tunicate Aplidium albicans, was used. The effect of the drug on human colon cultured cell lines was tested using multiple approaches: proliferation studies, long term survival after three hours of exposure to DDB by means of a clonogenic assay and the decrease of the protooncogen, ornithine decarboxylase, activity. A dehydrodidemnin B concentration of 10(-8) M completely inhibited cell growth. The IC50 obtained using the MTT proliferation test, indicated that the most proliferative cell line (CT-2) was the most sensitive to the drug. Using a clonogenic assay a clear dose-response was obtained for the three cell lines used; HT-29 cell line showed the minimum survival after 3 hours of dehydrodidemnin B treatment. A dose-dependent decrease in ornithine decarboxylase activity was also observed in three cell lines assayed. The data presented indicate that the dehydrodidemnin B is a potent cytotoxic agent on rapidly dividing human colon cancer cells. PMID:9066673

  15. Transcription profiles of non-immortalized breast cancer cell lines

    PubMed Central

    Fernandez-Cobo, Mariana; Holland, James F; Pogo, Beatriz GT

    2006-01-01

    Background Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Methods Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. Results According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. Conclusion The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research. PMID

  16. Characterisation of thyroid medullary carcinoma TT cell line.

    PubMed

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid. PMID:9046062

  17. Pathomorphological changes in gills of fish fingerlings (Cirrhina mrigala) by linear alkyl benzene sulfonate

    SciTech Connect

    Misra, V.; Lal, H.; Chawla, G.; Viswanathan, P.N.

    1985-12-01

    Fish fingerlings (Cirrhina mrigala) exposed to 0.005 ppm (25% of LC50) concentration to detergents (linear alkyl benzene sulfonate) showed marked behavioral changes and distorted appearance of primary and secondary lamellae along with damage to gill epithelium under scanning electron microscopy at various magnifications. Mucosal cells of gills were found to secrete mucus showing primary reactions for membrane damage leading to dysfunction in respiration and osmoregulation.

  18. Characterization of a human ovarian teratocarcinoma-derived cell line.

    PubMed

    Zeuthen, J; Nørgaard, J O; Avner, P; Fellous, M; Wartiovaara, J; Vaheri, A; Rosén, A; Giovanella, B C

    1980-01-15

    A cell line (PA I), derived from human ovarian teratocarcinoma cells, was obtained by culturing ascitic fluid cells from a patient with recurrence of malignant ovarian teratoma. During early passages the cultured cells showed a variable morphology, a long doubling time, and a low plating efficiency (2%). After about 50 passages in vitro, a cell population which was more homogeneous and resembled embryonal carcinoma cells were obtained. These cells had a shorter doubling time (26 h), and increased plating efficiency (77%). The early-passage cells were aneuploid (P 24) whereas the late-passage cells had a normal diploid karyotype with one balanced translocation between chromosomes No. 15 and No. 20 (P 224). Details of the karyotype suggest that the cells are heterozygous, i.e. derived from a stage before the first meiotic division. One of the two X chromosomes were inactive, and the cells expressed HLA antigens (A28 and B12), and beta 2-microglobulin. Expression of F9 antigen, characteristic of two-cell and later preimplantation embryos, was absent, while expression of PCC4 antigen, expressed also by blastocysts, was present. This finding suggests that the line might express some embryonic characteristics. The PA I cell line maintained in monolayer cultures showed several characteristics of malignant cells. The proportion of malignant cells increased with successive passages in vitro. The late-passage cells represented a fairly homogenous population of malignant cells similar to embryonal carcinoma cells. Late-passage PA I cells, when seeded under conditions that prevented attachment of cells to the substratum, formed embryoid bodies consisting of an inner core of cells similar to embryonal carcinoma cells, surrounded by a rind of endoderm-like cells. These two cell layers were separated by a basement membrane-like structure containing fibronectin. The core embryonal carcinoma cells expressed high alkaline phosphatase activity whereas the endoderm-like cells had low

  19. Cell Line Modeling to Study Biomarker Panel in Prostate Cancer

    PubMed Central

    NickKholgh, Bita; Fang, Xiaolan; Winters, Shira M.; Raina, Anvi; Pandya, Komal S.; Gyabaah, Kenneth; Fino, Nora; Balaji, K.C.

    2016-01-01

    BACKGROUND African–American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS We assembled a PCa cell line model that included currently available African–American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS The dysregulation of the multiplex biomarker panel in primary African–American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African–American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African–Americans and Caucasians as a prelude to future translational studies. CONCLUSION We have characterized a novel in

  20. 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.

  1. Measurement of Acetylcholine from Cell Lines

    PubMed Central

    Lau, Jamie K.; Brown, Kathleen C.; Dasgupta, Piyali

    2016-01-01

    Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80–90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.

  2. Steroid hormone secretion in inflammatory breast cancer cell lines.

    PubMed

    Illera, Juan Carlos; Caceres, Sara; Peña, Laura; de Andres, Paloma J; Monsalve, Beatriz; Illera, Maria J; Woodward, Wendy A; Reuben, James M; Silvan, Gema

    2015-12-01

    Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17β-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were <10%. The mean recovery rate of hormone added to the culture medium was >90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies. PMID:26495931

  3. Embryonic germ cell lines and their derivation from mouse primordial germ cells.

    PubMed

    Labosky, P A; Barlow, D P; Hogan, B L

    1994-01-01

    When primordial germ cells of the mouse are cultured on feeder layers with the addition of the polypeptide signalling molecules leukaemia inhibitory factor, Steel factor and basic fibroblast growth factor they give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells (EG cells) can be induced to differentiate extensively in culture and also form teratocarcinomas when injected into nude mice. Additionally, they contribute to chimeras when injected into host blastocysts. We have derived multiple EG cell lines from 8.5 days post coitum (dpc) embryos of C57BL/6 inbred mice. Four independent EG cell lines with normal male karyotypes have formed chimeras (up to 70% coat colour chimerism) when injected into BALB/c host blastocysts. Chimeric mice from all four cell lines are fertile, but only those from one line have transmitted coat colour markers through the germline. Studies have also been carried out to determine whether gonadal primordial germ cells can give rise to pluripotent EG cells. Germ cells from gonads of 15.5 dpc C57BL/6 embryos and newborn mice failed to produce EG cell lines. EG cell lines capable of forming teratocarcinomas and coat colour chimeras have been established from primordial germ cells of 12.5 dpc genital ridges. We are currently testing the genomic imprinting status of the insulin-like growth factor type 2 receptor gene (Igf2r) in our different EG cell lines. PMID:7835148

  4. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    EPA Science Inventory

    We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

  5. 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

  6. Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

    PubMed Central

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

    2009-01-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients. PMID:18192229

  7. Ultrastructural observations on feeding appendages and gills of Alvinella pompejana (Annelida, Polychaeta)

    NASA Astrophysics Data System (ADS)

    Storch, V.; Gaill, F.

    1986-09-01

    The feeding appendages of Alvinella pompejana obtained from a deep-sea hydrothermal vent environment are described. They are characterized by a ciliated groove, the cells of which have a very distinctive ultrastructure, by groups of bipolar receptor cells and by several kinds of gland cells. Among these, one cell type is in an upside down position suggesting a function completely different from other epidermal secretory cells. The gills differ considerably from the feeding appendages on the basis of their ultrastructure. Their epidermis is very irregular in height; basal infoldings give the blood access to a space coming very near to the external medium. The blood vascular system is open. On the other hand, the gills of Amphicteis gunneri are not effective sites of gas exchange, since their columnar epithelium is underlain with muscle cells. The cells composing the feeding appendages and gills of Alvinella pompejana are characterized by ultrastructurally very different mitochondria.

  8. Salmon Gill Poxvirus, the Deepest Representative of the Chordopoxvirinae

    PubMed Central

    Yutin, Natalya; Tengs, Torstein; Senkevich, Tania; Koonin, Eugene; Rønning, Hans Petter; Alarcon, Marta; Ylving, Sonja; Lie, Kai-Inge; Saure, Britt; Tran, Linh; Dale, Ole Bendik

    2015-01-01

    ABSTRACT Poxviruses are large DNA viruses of vertebrates and insects causing disease in many animal species, including reptiles, birds, and mammals. Although poxvirus-like particles were detected in diseased farmed koi carp, ayu, and Atlantic salmon, their genetic relationships to poxviruses were not established. Here, we provide the first genome sequence of a fish poxvirus, which was isolated from farmed Atlantic salmon. In the present study, we used quantitative PCR and immunohistochemistry to determine aspects of salmon gill poxvirus disease, which are described here. The gill was the main target organ where immature and mature poxvirus particles were detected. The particles were detected in detaching, apoptotic respiratory epithelial cells preceding clinical disease in the form of lethargy, respiratory distress, and mortality. In moribund salmon, blocking of gas exchange would likely be caused by the adherence of respiratory lamellae and epithelial proliferation obstructing respiratory surfaces. The virus was not found in healthy salmon or in control fish with gill disease without apoptotic cells, although transmission remains to be demonstrated. PCR of archival tissue confirmed virus infection in 14 cases with gill apoptosis in Norway starting from 1995. Phylogenomic analyses showed that the fish poxvirus is the deepest available branch of chordopoxviruses. The virus genome encompasses most key chordopoxvirus genes that are required for genome replication and expression, although the gene order is substantially different from that in other chordopoxviruses. Nevertheless, many highly conserved chordopoxvirus genes involved in viral membrane biogenesis or virus-host interactions are missing. Instead, the salmon poxvirus carries numerous genes encoding unknown proteins, many of which have low sequence complexity and contain simple repeats suggestive of intrinsic disorder or distinct protein structures. IMPORTANCE Aquaculture is an increasingly important global

  9. Germ line development: lessons learned from pluripotent stem cells.

    PubMed

    Martínez-Arroyo, Ana M; Medrano, Jose V; Remohí, José; Simón, Carlos

    2014-10-01

    Current knowledge about mammalian germ line development is mainly based on the mouse model and little is known about how this fundamental process occurs in humans. This review summarizes our current knowledge of genetic and epigenetic germ line development in mammals, mainly focusing on primordial germ cell (PGC) specification events, comparing the differences between mouse and human models. We also emphasize the knowledge derived from the most successful strategies used to generate germ cell-like cells in vitro in both models and major obstacles to obtaining bona fide in vitro-derived gametes are considered. PMID:25461452

  10. Derivation of human embryonic stem cell line Genea019.

    PubMed

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

    2016-03-01

    The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346002

  11. 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

    2014-09-01

    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

  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. Female Sex Bias in Human Embryonic Stem Cell Lines

    PubMed Central

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat

    2012-01-01

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  14. Female sex bias in human embryonic stem cell lines.

    PubMed

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat; Eiges, Rachel

    2012-02-10

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  15. Reynolds number effects on gill pumping mechanics in mayfly nymphs

    NASA Astrophysics Data System (ADS)

    Sensenig, Andrew; Shultz, Jeffrey; Kiger, Ken

    2006-11-01

    Mayfly nymphs have an entirely aquatic life stage in which they frequently inhabit stagnant water. Nymphs have the capability to generate a ventilation current to compensate for the low oxygen level of the water by beating two linear arrays of plate-like gills that typically line the lateral edge of the abdomen. The characteristic Reynolds number associated with the gill motion changes with animal size, varying over a span of Re = 5 to 100 depending on age and species. The assumption that the system maintains optimal energetic efficiency leads to the prediction that animals transition from rowing to flapping mechanisms with increasing Re, while possibly utilizing a squeeze mechanism to a greater extent at lower Re. To investigate this hypothesis, we capture the motion of the gills through 3D imaging to investigate the effect of Reynolds number on the stroke patterns. PIV is utilized to assess flow rates and viscous dissipation. The effectiveness of the ventilation mechanism at each size has important consequences for the range of oxygen levels, and hence the habitat range, that can be tolerated by that size.

  16. Generation of Mouse iNKT Cell Lines

    PubMed Central

    Li, Xiangming; Tsuji, Moriya; Schneck, Jonathan; Webb, Tonya J.

    2016-01-01

    Natural killer T (NKT) cells bridge the innate and adaptive arms of the immune system, and manipulating their effector functions can have therapeutic significances in the treatment of autoimmunity, transplant biology, infectious disease and cancer. This important lymphocyte subset regulates the immune system through their potent cytokine production following the recognition of lipid antigen present in the context of the MHC class I-like CD1d molecule, in addition their ability to directly mediate cytotoxicity. Here, we describe a method of expanding mouse invariant NKT (iNKT) cell lines from mononuclear cells isolated from the thymus, spleen, or liver using bone marrow derived dendritic cells. These iNKT cell lines can be used study their co-signaling requirements, cytokine profiles and cytotoxic functions which will greatly enhance our knowledge of iNKT cell biology.

  17. JNK Inhibition Inhibits Lateral Line Neuromast Hair Cell Development

    PubMed Central

    Cai, Chengfu; Lin, Jinchao; Sun, Shaoyang; He, Yingzi

    2016-01-01

    JNK signaling is known to play a role in regulating cell behaviors such as cell cycle progression, cell proliferation, and apoptosis, and recent studies have suggested important roles for JNK signaling in embryonic development. However, the precise function of JNK signaling in hair cell development remains poorly studied. In this study, we used the small molecule JNK inhibitor SP600125 to examine the effect of JNK signaling abrogation on the development of hair cells in the zebrafish lateral line neuromast. Our results showed that SP600125 reduced the numbers of both hair cells and supporting cells in neuromasts during larval development in a dose-dependent manner. Additionally, JNK inhibition strongly inhibited the proliferation of neuromast cells, which likely explains the decrease in the number of differentiated hair cells in inhibitor-treated larvae. Furthermore, western blot and in situ analysis showed that JNK inhibition induced cell cycle arrest through induction of p21 expression. We also showed that SP600125 induced cell death in developing neuromasts as measured by cleaved caspase-3 immunohistochemistry, and this was accompanied with an induction of p53 gene expression. Together these results indicate that JNK might be an important regulator in the development of hair cells in the lateral line in zebrafish by controlling both cell cycle progression and apoptosis. PMID:26903805

  18. Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

    PubMed Central

    Crameri, Gary; Todd, Shawn; Grimley, Samantha; McEachern, Jennifer A.; Marsh, Glenn A.; Smith, Craig; Tachedjian, Mary; De Jong, Carol; Virtue, Elena R.; Yu, Meng; Bulach, Dieter; Liu, Jun-Ping; Michalski, Wojtek P.; Middleton, Deborah; Field, Hume E.; Wang, Lin-Fa

    2009-01-01

    Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study. PMID:20011515

  19. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

    PubMed Central

    Cherbas, Lucy; Hackney, Jennifer; Gong, Lei; Salzer, Claire; Mauser, Eric; Zhang, Dayu; Cherbas, Peter

    2015-01-01

    We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. PMID:26450921

  20. 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

  1. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  2. Artificial islets from hybrid spheroids of three pancreatic cell lines.

    PubMed

    Jo, Y H; Jang, I J; Nemeno, J G; Lee, S; Kim, B Y; Nam, B M; Yang, W; Lee, K M; Kim, H; Takebe, T; Kim, Y S; Lee, J I

    2014-05-01

    Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, β cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a β-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development. PMID:24815150

  3. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

    Vergara, D; Simeone, P; De Matteis, S; Carloni, S; Lanuti, P; Marchisio, M; Miscia, S; Rizzello, A; Napolitano, R; Agostinelli, C; Maffia, M

    2016-01-01

    Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies. PMID:26588820

  4. Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

    1989-06-01

    The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

  5. Skin Biopsy and Patient-Specific Stem Cell Lines

    PubMed Central

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  6. Structural elements of the gills of the shore crab Carcinus mediterraneus

    NASA Astrophysics Data System (ADS)

    Venezia, L. Dalla; Zago, C.; Siebers, D.; Menetto, A.

    1992-12-01

    Fine structural studies were conducted on the gills of the shore crab Carcinus mediterraneus using scanning electron microscopic techniques. The results obtained show the structural organization of crab gills from whole gills including spiny elements over the 150 lamellae to lamellar components such as cuticles, median shaft, marginal canal, afferent and efferent lamellar vessels and hemolymph cells. Enormous surface enlargement is accomplished by a variety of structural elements which allow rapid circulation of hemolymph. In the form of a relatively small organ, the gills fulfill all the necessary exchanges of specific molecules between the crab and its environment. Aggregations of ca 1-μm particles covering the outer cuticular surfaces are considered to be bacterial colonies of unknown properties and functions.

  7. Intracellular Oceanospirillales bacteria inhabit gills of Acesta bivalves.

    PubMed

    Jensen, Sigmund; Duperron, Sébastien; Birkeland, Nils-Kåre; Hovland, Martin

    2010-12-01

    A novel bacterium was discovered in the gills of the large bivalve Acesta excavata (Limidae) from coral reefs on the northeast Atlantic margin near the shelf break of the fishing ground Haltenbanken of Norway, and confirmed present in A. excavata from a rock-wall in the Trondheimsfjord. Purified gill DNA contained one dominant bacterial rRNA operon as indicated from analysis of broad range bacterial PCR amplicons in denaturant gradient gels, in clone libraries and by direct sequencing. The sequences originated from an unknown member of the order Oceanospirillales and its 16S rRNA gene fell within a clade of strictly marine invertebrate-associated Gammaproteobacteria. Visual inspection by fluorescent in situ hybridization and transmission electron microscopy indicated a pleomorphic bacterium with no visible cell wall, located in aggregates inside vacuoles scattered within the gill cells cytoplasm. Intracellular Oceanospirillales exist in bathymodiolin mussels (parasites), Osedax worms and whiteflies (symbionts). This bacterium apparently lives in a specific association with the Acesta. PMID:21044098

  8. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    SciTech Connect

    Medina, D.; Oborn, C.J. ); Li, M.L.; Bissell, M.J. )

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  9. Characterization of a Novel Radiation-Induced Sarcoma Cell Line

    PubMed Central

    Lang, J.E.; Zhu, W.; Nokes, B.T.; Sheth, G.R.; Novak, P.; Fuchs, L.; Watts, G.S.; Futscher, B.W.; Mineyev, N.; Ring, A.; LeBeau, L.; Nagle, R.; Cranmer, L.D.

    2014-01-01

    Background Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. Methods We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. Results Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. Conclusion Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS. Synopsis We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a radiation-induced sarcoma (RIS). Our novel RIS cell line constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. PMID:25644184

  10. Human cell lines: A promising alternative for recombinant FIX production.

    PubMed

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins. PMID:26802680

  11. 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.

  12. Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch.

    PubMed

    Fines, G A; Ballantyne, J S; Wright, P A

    2001-01-01

    In elasmobranch fishes, urea occurs at high concentrations (350-600 mM) in the body fluids and tissues, where it plays an important role in osmoregulation. Retention of urea by the gill against this huge blood-to-water diffusion gradient requires specialized adaptations to the epithelial cell membranes. Experiments were performed to determine the mechanisms and structural features that facilitate urea retention by the gill of the spiny dogfish Squalus acanthias. Analysis of urea uptake by gill basolateral membrane vesicles revealed the presence of a phloretin-sensitive (half inhibition 0.09 mM), sodium-coupled, secondary active urea transporter (Michaelis constant = 10.1 mM, maximal velocity = 0.34 micromol. h(-1). mg protein(-1)). We propose that this system actively transports urea out of the gill epithelial cells back into the blood against the urea concentration gradient. Lipid analyses of the basolateral membrane revealed high levels of cholesterol contributing to the highest reported cholesterol-to-phospholipid molar ratio (3.68). This unique combination of active urea transport and modification of the phospholipid bilayer membrane is responsible for decreasing the gill permeability to urea and facilitating urea retention by the gill of Squalus acanthias. PMID:11124129

  13. 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).

  14. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

    PubMed Central

    2010-01-01

    Background The treatment of oral squamous cell carcinomas (OSCC) and human osteosarcoma (HOS) includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang) on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20%) were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50) for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis. PMID:20840769

  15. Simultaneous measurement of natural killer cell cytotoxicity against each of three different target cell lines.

    PubMed

    Blomberg, K

    1994-02-10

    A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium. PMID:8308301

  16. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

    Zhang, Yange; Huang, Wen; Ornelles, David A; Gooding, Linda R

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  17. Registration of Human Embryonic Stem Cell Lines: Korea, 2010

    PubMed Central

    Lee, Ji-Yoon; Lee, Dae-Yeon; Choi, Young-Sil; Lee, Kyoung-Jae; Kim, Yong-Ou

    2011-01-01

    In an effort to increase the credibility of human embryonic stem cell (hESC) lines established in Korea, obligatory registration was introduced by the Bioethics and Safety Act 2008, effective as of January 1, 2010. The DNA fingerprint, chromosome stability, expression of pluripotency markers, and contamination of mycoplasma of the submitted lines were analyzed by Korea Centers for Disease Control and Prevention (KCDC). The characterization data and ethical aspects, such as informed consent for donation of surplus embryos, were reviewed by a 10-member advisory review committee for stem cell registry. A total of 55 domestic hESC lines were submitted for registration in 2010; among them 51 were registered. Among these submitted lines, 26 were additionally characterized by KCDC, while 25 lines previously characterized by the Ministry of Education, Science and Technology were not additionally analyzed by KCDC. Registration completed an oversight system for embryo research by registering the products of licensed embryo research projects, making embryo research more transparent in Korea. Information about hESC lines is available at the website of the Korea Stem Cell Registry (kscr.nih.go.kr). PMID:24159464

  18. Gilles vs Pugh and the lost letter.

    PubMed

    Paull, J D

    2009-07-01

    In June 1842 the wife of Lewis W Gilles, a banker in Launceston, wrote to Mrs Pugh, inviting her and her husband, Mr William Russ Pugh, to an entertainment and ball. The Pughs neither responded to the invitation, nor attended the ball. Mrs Gilles mentioned the discourtesy of their non-attendance to a friend, who the following morning brought the complaint to the attention of Dr Pugh. Pugh immediately wrote a brief note explaining that neither he nor his wife had received the invitation. Further correspondence between Gilles and Pugh ensued, becoming more and more bitter until Pugh publicly insulted Gilles. The Launceston Club, of which both men were members, ultimately became a casualty of the dispute and was dissolved. A Supreme Court defamation and libel case followed with Gilles as plaintiff and Pugh as defendant. Gilles won the case but received only token damages. And the missing letter? Too late, Mrs Pugh's maid found it tucked between the pages of a periodical on the dresser PMID:19705633

  19. Expression of the somatostatin gene in human astrocytoma cell lines.

    PubMed Central

    Mercure, L; Tannenbaum, G S; Schipper, H M; Phaneuf, D; Wainberg, M A

    1996-01-01

    Somatostatin (somatotropin release-inhibiting hormone; SRIH) has been demonstrated in neurons of the central nervous system (CNS) as well as in endocrine cells of the pancreas and gastrointestinal tract and can suppress various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production. Since astrocytes possess antigen-presenting activity and can secrete a wide array of immunoregulatory and inflammatory cytokines, we studied SRIH gene expression in both astrocyte cell lines and mitogen-stimulated peripheral blood mononuclear leukocytes from healthy donors. We now report by means of a complementary DNA-based reverse transcription PCR that differential levels of SRIH mRNA were expressed in 9 of 11 human astrocytoma cell lines tested but were undetectable in activated peripheral blood mononuclear leukocytes as well as in a variety of human lymphocyte and monocyte cell lines. The synthesis and secretion of SRIH protein by astrocytoma cells that expressed SRIH transcripts were confirmed by specific radioimmunoassay of cell culture fluids. These findings support the notion that SRIH gene expression occurs in human astrocytoma cells but not in mature lymphoid cells of the immune system. PMID:8991628

  20. Molecular cytogenetic analysis of breast cancer cell lines

    PubMed Central

    Davidson, J M; Gorringe, K L; Chin, S-F; Orsetti, B; Besret, C; Courtay-Cahen, C; Roberts, I; Theillet, C; Caldas, C; Edwards, P A W

    2000-01-01

    The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/~pawefish. © 2000 Cancer Research Campaign PMID:11044355

  1. 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

  2. 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. PMID:27103160

  3. Derivation of ductlike cell lines from a transplantable acinar cell carcinoma of the rat pancreas.

    PubMed Central

    Pettengill, O. S.; Faris, R. A.; Bell, R. H.; Kuhlmann, E. T.; Longnecker, D. S.

    1993-01-01

    Two cell lines were derived from a transplantable acinar cell carcinoma that had been established from a primary carcinoma of the pancreas in an azaserine-treated Lewis rat. The cultured tumor cells initially produced amylase, but production of exocrine enzymes ceased after 1-2 weeks in culture. The cultured cells were tumorigenic in Lewis rats, and one line produced solid tumors composed of ductlike structures surrounded by dense fibrous tissue. The second cell line produced partially solid and partially cystic tumors with a mixed phenotype of squamous, mucinous, and glandular areas when it grew in vivo following regrafting. Both cell lines lost structural and immunohistochemical acinar cell markers while acquiring duct cell markers during culture and regrafting. These studies provide strong support for the hypothesis that ductlike carcinomas can arise from neoplastic pancreatic acinar cells in rats. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:8391218

  4. Generation of BAC reporter cell lines for drug discovery.

    PubMed

    Kao, Betty R; McColl, Bradley; Vadolas, Jim

    2015-01-01

    Bacterial artificial chromosome (BAC) reporter cell lines are generated through stable transfection of a BAC reporter construct wherein the gene of interest is tagged with a reporter gene such as eGFP. The large capacity of BACs (up to 350 kb of genomic sequence) enables the inclusion of all regulatory elements that ensure appropriate regulation of the gene of interest. Furthermore, the reporter gene allows the expression of the gene of interest to be readily detected by flow cytometry. Cell lines can also be easily cultured for extended periods with minimal cost. These features of BAC reporter cell lines make them highly amenable for use in high-throughput screening of large drug libraries for compounds that induce the expression of the gene of interest. This chapter describes a method for generation of BAC reporter cell lines that are suitable as cellular assay systems in high-throughput screening. Briefly, this method involves (A) generation of cell clones stably transfected with a BAC reporter construct, (B) selection of "candidate" cell clones based on the responsiveness to known inducers, (C) confirmation of the integrity of the BAC reporter construct integrated within the candidate clones, and (D) assessment of the developmental regulation of the BAC reporter construct. As an example, we describe the generation of a BAC reporter cell line containing the human β-globin locus modified to express γ-globin as eGFP for use as a cellular reporter assay for screening of drugs that can reactivate expression of developmentally silenced γ-globin for the treatment of β-hemoglobin disorders. PMID:25239756

  5. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  6. Cytolytic activity against tumor cells by macrophage cell lines and augmentation by macrophage stimulants.

    PubMed

    Taniyama, T; Holden, H T

    1980-07-15

    Previous studies have shown that macrophage cell lines retained the ability to phagocytize, to secrete lysosomal enzymes, and to function as effector cells in antibody-dependent cellular cytoxicity. In this paper, the cytolytic activity of murine macrophage cell lines against tumor target cells was assessed using an 18-h 51Cr release assay. Of the macrophage cell lines tested, RAW 264, PU5-1.8 and IC-21 had intermediate to high levels of spontaneous cytolytic activity, P388D, and J774 had low to intermediate levels, while /WEHI-3 showed little or no cytolytic activity against RBL-5, MBL-2 and TU-5 target cells. Tumor-cell killing by macrophage cell lines could be augmented by the addition of macrophage stimulants, such as bacterial lipopolysaccharide and poly I:C, indicating that the activation of macrophages by these stimulants does not require the participation of other cell types. Treatment with interferon also augmented the tumor-cell killing by macrophage cell lines. Although the mechanism by which these cell lines exert their spontaneous or boosted cytotoxic activity is not clear, it does not appear to be due to depletion of nutrients since cell lines with high metabolic and proliferative activities, such as WEHI-3 and RBL-5, showed little or no cytotoxicity and supernatants from the macrophage cell lines did not exert any cytotoxic effects in their essay. Thus, it appears that the different macrophage cell lines represent different levels of activation and/or differentiation and may be useful for studying the development of these processes as well as providing a useful tool for analyzing the mechanisms of macrophage-mediated cytolysis. PMID:6165690

  7. Genetic instability in human ovarian cancer cell lines.

    PubMed Central

    Orth, K; Hung, J; Gazdar, A; Bowcock, A; Mathis, J M; Sambrook, J

    1994-01-01

    We have analyzed the stability of microsatellites in cell lines derived from human ovarian cancers and found that 5 out of 10 of the ovarian tumor cell lines are genetically unstable at the majority of the loci analyzed. In clones and subclones derived serially from one of these cell lines (2774; serous cystadenocarcinoma), a very high proportion of microsatellites distributed in many different regions of the genome change their size in a mercurial fashion. We conclude that genomic instability in ovarian tumors is a dynamic and ongoing process whose high frequency may have been previously underestimated by PCR-based allelotyping of bulk tumor tissue. We have identified the source of the genetic instability in one ovarian tumor as a point mutation (R524P) in the human mismatch-repair gene MSH2 (Salmonella MutS homologue), which has recently been shown to be involved in hereditary nonpolyposis colorectal cancer. Patient 2774 was a 38-year-old heterozygote, and her normal tissue carried both mutant and wild-type alleles of the human MSH2 gene. However the wild-type allele was lost at some point early during tumorigenesis so that DNA isolated either from the patient's ovarian tumor or from the 2774 cell line carries only the mutant allele of the human MSH2 gene. The genetic instability observed in the tumor and cell line DNA, together with the germ-line mutation in a mismatch-repair gene, suggest that the MSH2 gene is involved in the onset and/or progression in a subset of ovarian cancer. Images PMID:7937795

  8. Hypoxic cell turnover in different solid tumor lines

    SciTech Connect

    Ljungkvist, Anna S.E. . E-mail: a.ljungkvist@rther.umcn.nl; Bussink, Johan; Kaanders, Johannes H.A.M.; Rijken, Paulus F.J.W.; Begg, Adrian C.; Raleigh, James A.; Kogel, Albert J. van der

    2005-07-15

    Purpose: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. Methods and Materials: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). Results: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. Conclusions: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

  9. 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.

  10. Cell membrane fatty acid composition differs between normal and malignant cell lines.

    PubMed

    Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska

    2004-06-01

    Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

  11. Acidophilic granulocytes in the gills of gilthead seabream Sparus aurata: evidence for their responses to a natural infection by a copepod ectoparasite.

    PubMed

    Lui, Alice; Manera, Maurizio; Giari, Luisa; Mulero, Victoriano; Dezfuli, Bahram Sayyaf

    2013-09-01

    Immunohistochemical and ultrastructural studies were conducted on the gills of gilthead seabream, Sparus aurata L., naturally infected with the copepod ectoparasite Ergasilus lizae (Krøyer, 1863) in order to assess pathology and the host immune cell response. Gills of 56 gilthead seabream were screened for ectoparasites; 36 specimens (64.3%) harbored E. lizae. Intensity of infection was 32.7 ± 8.7 (mean ± SE). Pathological alterations to the gills of the host were more pronounced in close proximity to the copepod site of attachment. The parasite attached to the gills by means of its modified second antennae, occluded the arteries, provoked epithelial hyperplasia and hemorrhages and most often caused lamellar disruption. Numerous granular cells were encountered near the site of E. lizae attachment. In both infected and uninfected gills, the granular cells lay within the filaments and frequently occurred within the connective tissue inside and outside the blood vessels of the filaments. The type of granular cell was identified by immunohistochemical staining by using the monoclonal antibody G7 (mAb G7), which specifically recognizes acidophilic granulocytes (AGs) of S. aurata and with an anti-histamine antibody (as a marker for mast cells, MCs) on sections from 13 uninfected gills and 21 parasitized gills. The use of mAb G7 revealed that, in gills harboring copepods, the number of G7-positive cells (i.e., AGs; 32.9 ± 3.9, mean number of cells per 45,000 μm2 ± SE) was significantly higher than the density of the same cells in uninfected gills (15.3 ± 3.8; ANOVA, P < 0.05). Few histamine-positive granular cells (i.e., MCs) were found in the uninfected and parasitized gills. Here, we show, for the first time in S. aurata infected gills, that AGs rather than MCs are recruited and involved in the response to E. lizae infection in seabream. PMID:23644766

  12. Establishing an immortalized human osteoprecursor cell line: OPC1.

    PubMed

    Winn, S R; Randolph, G; Uludag, H; Wong, S C; Hair, G A; Hollinger, J O

    1999-10-01

    The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P

  13. Derivation of Ethnically Diverse Human Induced Pluripotent Stem Cell Lines.

    PubMed

    Chang, Eun Ah; Tomov, Martin L; Suhr, Steven T; Luo, Jiesi; Olmsted, Zachary T; Paluh, Janet L; Cibelli, Jose

    2015-01-01

    The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair, and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian, and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations. PMID:26482195

  14. Establishment and applications of male germ cell and Sertoli cell lines.

    PubMed

    Wang, Hong; Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; He, Zuping

    2016-08-01

    Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine. PMID:27069011

  15. Detection of cytoplasmic and surface membrane markers in cells of some human hematopoietic cell lines.

    PubMed

    Koníková, E; Babusíková, O; Kusenda, J; Glasová, M

    1992-01-01

    The cells of some human leukemia-lymphoma T cell lines (JURKAT, MOLT4), B cell lines (DAUDI, U-266) and of myeloid U-937 cell line were characterized for their surface membrane and cytoplasmic marker profiles. The usefulness of some fixation and permeabilization methods of cell membrane for detection of cytoplasmic markers by flow cytometry was studied. The methods of cell fixation in suspension were found to be more sensitive than the methods of cell fixation in smears. With the very short buffered formaldehyde-acetone (BFA) fixation used in this study an optimal penetration of the monoclonal antibodies (MoAbs) through the plasma membrane and specific binding to the appropriate structures were achieved. CD22 antigen was detected in cytoplasm but not on membrane of DAUDI cells. In another B cell line, U-266, CD22 antigen was present both in cell membrane and cytoplasm. The marker corresponding to anti-CD19 MoAb was detected in cytoplasm but was absent on membrane of U-266 cells. Furthermore, the antigen estimated by anti-CD3 MoAb could be detected intracellularly in cells of both T cell lines tested, while it was absent on cell membrane of these cells. The phenotypic study of U-937 cells showed that the majority of cells expressed myeloid associated antigens. In our study the CD14 marker detected on cell surface membrane of U-937 cells was missing in their cytoplasm. The surface antigens remained intact after BFA fixation enabling a simultaneous detection of membrane and cytoplasmic markers in double immunofluorescence studies. Through this combination of markers minor cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for a rapid and quantitative immunodiagnosis. PMID:1491722

  16. 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

  17. Transcriptional profiling and assessment of cell lines as in vitro models for mantle cell lymphoma.

    PubMed

    Ek, Sara; Ortega, Eva; Borrebaeck, Carl A K

    2005-02-01

    Mantle cell lymphoma (MCL) is an aggressive malignancy and new treatment modalities must be established to increase patient survival time. In the search for new therapeutic targets, reliable and well-characterised in vitro models are essential. In this study, we have characterised three MCL cell lines (SP53, Granta 519 and NCEB1) in comparison with primary tumours from MCL, follicular lymphomas (FL), a FL cell line (RL), a Burkitt lymphoma cell line (RAJI) and five different B cell populations from healthy individuals. Expression profiling was used to determine the relative expression of >12000 transcripts in these samples, and flow cytometry analysis was performed to establish a phenotypic signature for each of the cell lines. In addition, the cell lines were sequenced, and the frequency of somatic mutations and immunoglobulin (Ig) variable heavy chain (VH) usage were determined. We show by hierarchical clustering that the cell lines retain a genetic signature similar to primary MCL, which readily separated the MCL samples from the other lymphoma cell lines and the FL tumours. Furthermore, the MCL cell lines showed differences in the frequency of VH somatic mutations (0-2.1%). The increased number of mutations in NCEB1, compared to the other MCL cell lines, was in agreement with a decreased expression of CD31, CD44, CXCR5, CCR7 and CCR6. Taken together, our data show that the cell lines are clearly derived from MCL tumours and expressed similar genetic and phenotypic signatures compared to primary tumours, which confirmed their usefulness as in vitro models. PMID:15607370

  18. Prevalence, site and tissue preference of myxozoan parasites infecting gills of cultured fish in Punjab (India).

    PubMed

    Kaur, Harpreet; Katoch, Anu

    2016-02-25

    Native carp species cultured in Indian farms in Punjab (catla Catla catla, rohu Labeo rohita, mrigal Cirrhinus mrigala, exotic carps such as silver carp Hypophthalmichthys molitrix, grass carp Ctenopharyngodon idella, common carp Cyprinus carpio and a catfish Sperata seenghala) were examined for the presence of myxozoan parasites infecting gills. Firstly, the gills were examined under a zoom-stereomicroscope for the presence of plasmodia. The number of plasmodia per gill was counted to determine the index for the intensity of infection. Infected tissues were processed for histology, and 3-4 µm sections of infected gills were stained with haematoxylin & eosin and Luna's method. A total of 19 species of myxosporean were found infecting various cell types in the gills. Of these, 14 species belonged to the genus Myxobolus, 3 species to the genus Thelohanellus and 2 species to the genus Henneguya. Species belonging to the genus Myxobolus formed the interlamellar and intralamellar vascular (LV) type plasmodia, and species belonging to the genus Thelohanellus and Henneguya formed intrafilamental vascular (FV) type plasmodia. Mixed infections comprising 2, 3 or 4 different myxozoan species were noted in individual fish. The most common type of parasitism was polyparasitism due to 4 myxobolids co-occuring in fish with an infection rate of 23.16%. All species caused mild to severe haemorrhagic gill disease with little clinical symptomatology. PMID:26912043

  19. Identification of Methanotrophic Biomarker Lipids in the Symbiont-Containing Gills of Seep Mussels

    NASA Technical Reports Server (NTRS)

    Jahnke, L. L.; Zahiralis, K. D.; Klein, H. P.; Morrison, David (Technical Monitor)

    1994-01-01

    Mussels collected from hydrocarbon seeps in the Gulf of Mexico grow with methane as sole carbon and energy source due to a symbiotic association with methane-oxidizing bacteria. Transmission electron micrographs of mussel gills show cells with stacked intracytoplasmic membranes similar to type I methanotrophic bacteria. Methanotrophs are known to synthesize several types of cyclic triterpenes, hopanoids and methyl sterols, as well as unique monounsaturated fatty acid, double bond positional isomers that serve as biomarkers for this group. Lipid analysis of dissected mussels demonstrated the presence of these biomarkers predominantly in the gill tissue with much smaller amounts in mantle and remaining body tissues. Gill tissue contained 1150 micrograms/g dry wt. of hopanepolyol derivatives and diplopterol while the mantle tissue contained only 17 micrograms/g. The C16 monounsaturated fatty acids (16:1) characteristic of type I methanotrophic membranes dominated the gill tissue making up 53% of the total while only 5% 16:1 was present in the mantle tissue. The methyl sterol distribution was more dispersed. The predominant sterol in all tissues was cholesterol with lesser amounts of other desmethyl and 4-methyl sterols. The gill and mantle tissues contained 3461 micrograms (17% methyl) and 2750 micrograms (5% methyl) sterol per gm dry wt., respectively. Methyl sterol accounted for 44% of the sterol esters isolated from the gill, suggesting active demethylation of the methanotrophic sterols in this tissue. The use of lipid biomarkers could provide an effective means for identifying host-symbiont relationships.

  20. [Establishment and biological characterization of human medulloblastoma cell lines].

    PubMed

    Yamada, M; Shimizu, K; Tamura, K; Okamoto, Y; Matsui, Y; Moriuchi, S; Park, K; Mabuchi, E; Yamamoto, K; Hayakawa, T

    1989-07-01

    Two cell lines of human medulloblastoma (ONS-76 and ONS-81) were established, and their biological characteristics were investigated. The cell line, ONS-76, was established from a tumor specimens obtained from a large cerebellar tumor of a 2-year-old girl. The pathological diagnosis was a typical medulloblastoma. The other cell line, ONS-81, was derived from a metastatic tumor in right frontal lobe of a 9-year-old girl. The tumor specimens were minced into fragments approximately 1 mm in diameter and cultured in plastic culture flasks in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50% patients serum. The cells growing as a monolayer were subcultured in RPMI 1640 supplemented with 10% FCS and initially with L-glutamine, sodium pyruvate, and nonessential amino acid. Microscopically, both cultured cells exhibited various morphological appearances, and this morphological heterogeneity seemed to be specific for medulloblastoma cells. The in vitro population doubling time of ONS-76 and ONS-81 were 18.6 and 19.2 hr, respectively. The ONS-76 and ONS-81 cells formed subcutaneous tumors in nude mice as serial transplantable xenograft, and these tumors had a microscopic appearance similar to that of the original medulloblastoma. Ultrastructurally++, the cultured cells showed primitive, undifferentiated appearance, and no neuronal or glial structures were not seen. Immunohistochemical studies showed that both cells expressed neuron-specific enolase (NSE) and neurofilament protein (NFP 200 K, 145 K), but glial fibrillary acidic protein (GFAP) and S-100 protein were not detected. The NFP immunoreactivities of both cultured cells were demonstrated as abnormal perinuclear deposits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2818910

  1. A Novel Cell Line from Spontaneously Immortalized Murine Microglia

    PubMed Central

    Kulas, Joshua; Combs, Colin K.

    2014-01-01

    Background Purified microglia cultures are useful tools to study microglial behavior in vitro. Microglial cell lines serve as an attractive alternative to primary microglia culture, circumventing the costly and lengthy preparation of the latter. However, immortalization by genetic or pharmacologic manipulations may show altered physiology from primary microglia. New Method A novel microglial cell line was isolated from a primary glial culture of postnatal murine cerebral cortices. The culture contained a population of spontaneously transformed microglia that continued to divide without genetic or pharmacological manipulations. After several clones were isolated, one particular clone, SIM-A9, was analyzed for its microglial characteristics. Results SIM-A9 cells expressed macrophage/microglia-specific proteins, CD68 and Iba1. SIM-A9 cells were responsive to exogenous inflammatory stimulation with lipopolysaccharide and β-amyloid, triggering tyrosine kinase-based and NFκB signaling cascades as well as TNFα secretion. SIM-A9 cells also exhibited phagocytic uptake of fluorescent labeled β-amyloid and bacterial bioparticles. Furthermore, lipopolysaccharide increased the levels of inducible nitric oxide synthase and cyclooxygenase-2, whereas IL-4 stimulation increased arginase-1 levels demonstrating that SIM-A9 cells are capable of switching their profiles to pro- or anti-inflammatory phenotypes, respectively. Comparison with Existing Methods The use of SIM-A9 cells avoids expensive and lengthy procedures required for the preparation of primary microglia. Spontaneously immortalized SIM-A9 cells are expected to behave more comparably to primary microglia than virally transformed or pharmacologically induced microglial cell lines. Conclusions SIM-A9 cells exhibit key characteristics of cultured primary microglia and may serve as a valuable model system for the investigation of microglial behavior in vitro. PMID:24975292

  2. 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...

  3. A human gall-bladder signet ring cell carcinoma cell line.

    PubMed

    Nishida, T; Iwasaki, H; Johzaki, H; Tanaka, S; Watanabe, R; Kikuchi, M

    1997-06-01

    To date, very few reports of the establishment of gall-bladder cancer cell lines have appeared, although many cancer cell lines of various kinds have been established. On the other hand, no reports could be found on signet ring cell carcinoma cell lines derived from the gall-bladder and only five cell lines from the stomach. A human gall-bladder cancer cell line (FU-GBC-2) was established in tissue culture from the ascitic fluid of a 69-year-old Japanese female patient. The tumor cells growing in tissue culture exhibited the morphological characteristics of signet ring cells in phase contrast and electron microscopy. The population doubling time was 43 hours. Heterotransplantation was succeeded by inoculation into the dermis of BALB/c nude mice. An immunocytochemical study showed that most of the cultured cells were positive for carcinoembryonic antigen, CA19-9 and epithelial membrane antigen, but negative for vimentin. The modal chromosome number was 120 with a range of 100-124. Flow cytometry showed an aneuploidy pattern in the cultured cells at passage 30. Markedly amplified c-myc oncogene was observed by Southern blot analysis. This cell line may be useful in the study of the morphological and biological characteristics of signet ring cell carcinoma and gall-bladder adenocarcinoma. PMID:9211524

  4. 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...

  5. 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. PMID:27099306

  6. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines.

    PubMed

    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

  7. DIFFERENCES IN ARACHIDONIC ACID METABOLISM BY HUMAN MYELOMONCYTIC CELL LINES

    EPA Science Inventory

    The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines were determined after incubation with interferongamma (IFNg; 500 U/ml) or vehicle for 4 days. ells were prelabeled with tritiated arachidonic acid for 4 hours, and media supernata...

  8. USING NEUROBLASTOMA CELL LINES TO EXAMINE ORGANOPHOSPHATE NEUROTOXICITY

    EPA Science Inventory

    The need to deploy IN VITRO models to test neurotoxic scribes the use of by industry and government regulatory agencies. his research describes the neuroblastoma cell lines to address the relationship between esterase inhibition and neurotoxic outcome following exposure to organo...

  9. 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...

  10. 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...