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Sample records for hamster embryo cells

  1. Cytological and oncogene alterations in radiation-transformed Syrian hamster embryo cells

    International Nuclear Information System (INIS)

    Trutschler, K.; Hieber, L.; Kellerer, A.M.

    1991-01-01

    Syrian hamster embryo (SHE) cells were neoplastically transformed by different types of ionizing radiation (γ-rays, α-particles or carbon ions). Transformed and tumor cell lines (derived from nude mice tumors) were analysed for alterations of the oncogenes c-Ha-ras and c-myc, i.e. RFLPs, gene amplifications, activation by point mutation, gene expression, and for cytological changes. In addition, the chromosome number and the numbers of micronuclei per cell have been determined in a series of cell lines. (author)

  2. No-Disjunction and loss of anafasica Hamster-human hybrid embryos of two cells

    International Nuclear Information System (INIS)

    Ponsa, I.; Tusell, L.; Alvarez, R.; Genesca, A.; Miro, R.; Egozcue, J.

    1998-01-01

    To investigate the possible effect anafasica the ionizing radiations in masculine germinal cells a new test it has been developed combining two techniques, the fecundation interspecific gives ovocitos hamster without area pellucid with human sperms and the fluorescent in situ hybridization in cells in interface using probes gives DNA specific centrometricas. Analyzing the segregation gives the chromosomes marked in the embryos two cells, you can detect the reciprocal products easily an anomalous segregation. Give this way the recount the fluorescent signs in the nuclei siblings and in the micronucleus it provides an esteem the due aneuploidy to errors meiotic or premiotic, with this way the resulting aneuploidy the errors in the first division mitotic the embryos, as much no-disjunction as lost anafasica

  3. Misoprostol-induced radioprotection of Syrian hamster embryo cells in utero from cell death and oncogenic transformation

    International Nuclear Information System (INIS)

    Miller, R.C.; LaNasa, P.; Hanson, W.R.

    1994-01-01

    Misoprostol, a PGE analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems. The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro. Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostal, and 2 h later the pregnant hamsters were exposed to graded doses of X rays. Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium. After a 2-week incubation period, clongenic cell survival and morphologically transformed foci were determined. Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone. In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone. These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events. 26 refs., 6 figs., 2 tabs

  4. Neoplastic transformation of hamster embryo cells irradiated in utero and assayed in vitro

    International Nuclear Information System (INIS)

    Borek, C.; Pain, C.; Mason, H.

    1977-01-01

    It is stated that induction of neoplastic transformation in vitro by x-rays and neutrons has been reported, and the authors had previously found that transformation by x-rays could be detected at doses as low as 1 R and the rate of transformation increased with dose, reaching a peak of 1% between 150 and 300 R. This frequency of neoplastic transformation in vitro is much higher than the frequency of radiation induced tumors observed after exposing animals to similar doses of radiation. Studies are here reported showing that malignant transformed cells can be obtained from embryos irradiated in utero and assayed in vitro, and that the frequency of transformation is at least tenfold lower than when the irradiations are performed in vitro, and thus closer to the incidence in animals. Hamster embryo cells were used for the studies. Questions that arise are as follows: does the host mediate in modulating transformation by radiation; is there a repair of transforming events before they can be expressed; and how significant is the state of cells during irradiation in determining the rate of transformation. It is known from in vitro studies that cell replication is required for fixation of the transformation. With the in vitro technique cells are seeded as single cells with ample opportunity to divide. In addition they are not in contact with one another, and constitute a mixture of cell types from many tissues. In utero the situation is quite different; the embryonic cells are irradiated as tissues where there is cell to cell contact in tissue-specific arrangements, and where the rate of cell replication varies with the tissue. It remains to be seen which of these factors, if any, is responsible for the lowered yield of transformed cells characteristic of in utero as opposed to in vitro irradiation. (U.K.)

  5. Gene amplification in Chinese hamster embryo cells by the decay of incorporated iodine-125

    International Nuclear Information System (INIS)

    Luecke-Huhle, Christine; Ehrfeld, Angelika; Rau, Waltraud

    1988-01-01

    Simian Virus 40-transformed Chinese hamster embryo cells (Co631) contain 5 viral copies integrated per cell genome. These SV40 sequences were used as an endogenous indicator gene to study response of mammalian cells to radiation at gene level. Cells were internally irradiated by Auger electrons emitted by Iodine-125 which was incorporated in cell DNA in form of 5-[ 125 I] iododeoxyuridine ( 125 IdU). An increase in gene copy number was measured using dispersed cell blotting and Southern analysis in combination with highly sensitive DNA hybridization. A 13-fold amplification of the SV40 sequences and a 2-fold amplification of two cellular oncogenes of the ras family were found. Other cellular genes, like the α-actin gene, are not amplified and no variation in gene copy number was observed after incubation of cells with cold IdU. Thus, specific gene amplification seems to be the consequence of radiation-induced DNA damage and the resulting cell cycle arrest. (author)

  6. Influence of dose rate on the transformation of Syrian hamster embryo cells by fission-spectrum neutrons

    International Nuclear Information System (INIS)

    Jones, C.A.; Sedita, B.A.; Hill, C.K.; Elkind, M.M.

    1988-01-01

    Several explanations for this neutron dose-rate effect have been proposed, but further investigation is necessary to determine the mechanisms involved. In all cell transformation studies to date the immortalized, aneuploid 10T1/2 cell-line has been used. These cells may be premalignant; thus their response characteristics and, in particular, the nature of the transformation event, might differ from that in a normal, fibroblast cell. One reason for the present study was to determine whether the low-dose-rate effect of fission neutrons could be demonstrated in normal cells. If so, a normal cell system, which would more closely resemble a normal in vivo system, could be used for mechanistic studies. We chose Syrian hamster embryo (SHE) fibroblasts which are normal, diploid cells with a limited life span in culture. Upon exposure to low doses of ionizing radiation, the fraction of the cells that are transformed can be identified in a standard 8--10 day colony assay by examining their clonal morphology. Transformed cells form colonies with a dense, criss-crossed or piled-up structure. A high percentage of the transformed colonies can be further propagated and will acquire additional neoplastic characteristics; i.e., anchorage independence, immortality, altered proteolytic activity, karyotype alterations, and finally, tumorigenicity

  7. Influence of dose rate on the transformation of Syrian hamster embryo cells by fission-spectrum neutrons

    Energy Technology Data Exchange (ETDEWEB)

    Jones, C.A.; Sedita, B.A.; Hill, C.K.; Elkind, M.M.

    1988-01-01

    Several explanations for this neutron dose-rate effect have been proposed, but further investigation is necessary to determine the mechanisms involved. In all cell transformation studies to date the immortalized, aneuploid 10T1/2 cell-line has been used. These cells may be premalignant; thus their response characteristics and, in particular, the nature of the transformation event, might differ from that in a normal, fibroblast cell. One reason for the present study was to determine whether the low-dose-rate effect of fission neutrons could be demonstrated in normal cells. If so, a normal cell system, which would more closely resemble a normal in vivo system, could be used for mechanistic studies. We chose Syrian hamster embryo (SHE) fibroblasts which are normal, diploid cells with a limited life span in culture. Upon exposure to low doses of ionizing radiation, the fraction of the cells that are transformed can be identified in a standard 8--10 day colony assay by examining their clonal morphology. Transformed cells form colonies with a dense, criss-crossed or piled-up structure. A high percentage of the transformed colonies can be further propagated and will acquire additional neoplastic characteristics; i.e., anchorage independence, immortality, altered proteolytic activity, karyotype alterations, and finally, tumorigenicity.

  8. The insecticide buprofezin induces morphological transformation and kinetochore-positive micronuclei in cultured Syrian hamster embryo cells in the absence of detectable DNA damage.

    Science.gov (United States)

    Herrera, L A; Ostrosky-Wegman, P; Schiffmann, D; Chen, Q Y; Ziegler-Skylakakis, K; Andrae, U

    1993-11-01

    The insecticide buprofezin was examined for its genotoxicity in cultured Syrian hamster embryo cells in order to better understand the mechanisms underlying the genotoxicity of the compound in mammalian cells. Exposure to buprofezin concentrations of 12.5-100 microM did not significantly affect the colony-forming ability of the cells, but did result in increased frequencies of morphologically transformed colonies. Treatment with buprofezin did not cause a detectable induction of DNA repair synthesis, an indicator of DNA damage, but significantly increased the frequency of micronuclei. Immunostaining of the cells with antikinetochore antibody (CREST antibody) showed that essentially all of the buprofezin-induced micronuclei were kinetochore-positive. The results suggest that morphological transformation of Syrian hamster embryo cells by buprofezin results from an interaction of the compound or a metabolite of it with the mitotic apparatus rather than from DNA damage.

  9. Promotion of TNF-α on malignant transformation of syrian hamster embryo cells irradiated with α-particles

    International Nuclear Information System (INIS)

    Zhu Maoxiang; Guo Renfeng; Yang Zhihua; GongYifen

    1999-01-01

    Objective: To illustrate the role of tumor necrosis factor-α (TNF-α) in radiation-induced cancer and regulatory mechanism of protein tyrosine phosphorylation. Methods: Taking Syrian hamster embryo cells exposed to 0.5 Gy α-particles as the target, an array of biological indicators such as cell growth curve, transformation frequency (TF), colony formation efficiency (CFE) and tumor formation in nude mice were observed, and the activities of protein tyrosine kinases and protein tyrosine phosphatases were measured. Results: Neither 0.5 Gy α-particle irradiation nor TNF-α alone could induce transformation of SHE cells morphologically, but the TF, CFE and levels of protein tyrosine phosphorylation were obviously increased in SHE cells treated with 600 U/ml TNF-α after exposure to 0.5 Gy α-particles, and malignant transformation was proved by tumorigenicity assays. Conclusion: TNF-α promotes significantly the transformation of SHE cells induced by α-particles, and protein tyrosine phosphorylation is probably involved in regulation of the process

  10. Multistep change in epidermal growth factor receptors during spontaneous neoplastic progression in Chinese hamster embryo fibroblasts

    International Nuclear Information System (INIS)

    Wakshull, E.; Kraemer, P.M.; Wharton, W.

    1985-01-01

    Whole Chinese hamster embryo lineages have been shown to undergo multistep spontaneous neoplastic progression during serial passage in culture. The authors have studied the binding, internalization, and degradation of 125 I-labeled epidermal growth factor at four different stages of transformation. The whole Chinese hamster embryo cells lost cell surface epidermal growth factor receptors gradually during the course of neoplastic progression until only 10% of the receptor number present in the early-passage cells (precrisis) were retained in the late-passage cells (tumorigenic). No differences in internalization rates, chloroquine sensitivity, or ability to degrade hormone between the various passage levels were seen. No evidence for the presence in conditioned medium of transforming growth factors which might mask or down-regulate epidermal growth factor receptor was obtained. These results suggest that a reduction in cell surface epidermal growth factor receptor might be an early event during spontaneous transformation in whole Chinese hamster embryo cells

  11. Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. I. Radical formation as studied by electron spin resonance

    International Nuclear Information System (INIS)

    Miyazaki, T.; Hayakawa, Y.; Suzuki, K.; Suzuki, M.; Watanabe, M.

    1990-01-01

    Formation of free radicals in golden hamster embryo (GHE) cells in the frozen living state by gamma irradiation has been studied by electron spin resonance spectroscopy at 4.2 and 77 K. The relative yields of H atoms, OH radicals, and organic radicals trapped in the irradiated GHE cells are 12, 72, and 16%, respectively, of total radical yields. When dimethylsulfoxide (DMSO) is added to GHE cells at 77 K, a large quantity of CH2SOCH3 radicals (DMSO radicals) are formed after gamma irradiation. The yields of OH radicals are not affected by the addition of DMSO. When the GHE cell-DMSO mixtures are irradiated with gamma rays at 77 K and then warmed to 111 K, the OH radicals decay, whereas the DMSO radicals do not increase complementarily. Moreover, the decay rates of the OH radicals at 111 K do not depend upon the concentration of DMSO. Thus OH radicals do not react with DMSO during warming of the irradiated sample. When H atoms are produced by gamma irradiation of acid ice at 60 K, the decay rates of the H atoms at 77 K increase with increasing DMSO concentration, indicating that DMSO reacts with H atoms (CH3SOCH3 + H----.CH2SOCH3 + H2) at 77 K by quantum-mechanical tunneling. When the GHE cell-DMSO mixture is irradiated with gamma rays at 77 or 4.2 K in the dark, DMSO ions are produced in addition to DMSO radicals. Therefore it is concluded that DMSO does not scavenge OH radicals, but does capture H atoms, holes and/or electrons in the gamma-irradiated cells, resulting in the remarkable formation of DMSO radicals. This scavenger effect of DMSO may be related to the radioprotection of DMSO against cell killing

  12. Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to γ-rays at 4 and 77 K as studied by electron spin resonance and biological measurements

    International Nuclear Information System (INIS)

    Miyazaki, T.; Suzuki, K.; Watanabe, M.

    1992-01-01

    Many studies have reported the biological effects of gamma-irradiation on cells. It has been generally accepted that OH radicals produced by radiolysis of water in cells play an important role in the biological effect. OH radicals, however, were not observed directly in these studies. Thus there is some ambiguity in the determination of the reactive species responsible for the biological effect. The effect of dimethyl sulfoxide on gamma-irradiated golden hamster embryo cells has been studied here at 4 and 77 K by direct observation of free radicals and by biological measurements. (author). 2 refs., 4 figs

  13. Alterations in the metabolism of benzo[a]pyrene in syrian hamster embryo (SHE) cells pretreated with phenolic antioxidants

    International Nuclear Information System (INIS)

    Strniste, G.F.; Okinaka, R.T.; Chen, D.J.

    1983-01-01

    Inhibition of chemical- or radiation-induced neoplasia has been observed in animals whose diets were supplemented with antioxidants commonly used as food additives. Inhibition of the carcinogenicity of benzo[a]pyrene (BaP) or of 7,12-dimenthylbenz[a]anthracene (DMBA) - in rats has been achieved by the addition of the phenolic antioxidants butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) to the diet. Our data suggest that in SHE cells antioxidants inhibit the overall metabolism of BaP to its various oxidized moieties including 7,8-diol- and 7,8,9,10-tetrol-BaP. A plausible explanation for our results with SHE cells is that the antioxidants interact directly with AHH, thus inhibiting AHH metabolic capacity. From analysis of nuclear material from SHE cells (+- antioxidants) incubated for 36 hours with BaP at 1 μg/ml, it is calculated that 4.6, 2.4 and 2.9 pmol BaP are bound to the DNA isolated from 10 7 nuclei of control, BHA-(20 μg/ml) and p-MP-(10 μg/ml) treated cultures, respectively

  14. Intracellular pH in increased after transformation of Chinese hamster embryo fibroblasts

    International Nuclear Information System (INIS)

    Ober, S.S.; Pardee, A.B.

    1987-01-01

    These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pH/sub i/) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. pH measurements were made with [ 14 C]-benzoic acid. This increase of pH/sub i/ in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na + /H + -antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pH/sub i/ regulation in the tumorigenic CHEF/18 derivatives lies in the Na + /H + antiporter itself. Further studies to determine the biological significance of an increased pH/sub i/ show that the external pH (pH 0 )-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pH/sub i/ in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pH/sub i/ can contribute to the acquisition of altered growth properties

  15. Proteomic Analysis of Chinese Hamster Ovary Cells

    DEFF Research Database (Denmark)

    Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama

    2012-01-01

    To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimens......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...

  16. Track segment studies with Chinese hamster cells

    International Nuclear Information System (INIS)

    Bird, R.P.

    1984-01-01

    Survival curves of near-diploid and near-tetraploid Chinese hamster cell cultures following irradiation by an 241 Am α source indicate different growth rates for the two clones. Possible reasons for the difference are discussed

  17. Methods for modeling chinese hamster ovary (cho) cell metabolism

    DEFF Research Database (Denmark)

    2015-01-01

    Embodiments of the present invention generally relate to the computational analysis and characterization biological networks at the cellular level in Chinese Hamster Ovary (CHO) cells. Based on computational methods utilizing a hamster reference genome, the invention provides methods for identify...

  18. Hamster thecal cells express muscle characteristics

    International Nuclear Information System (INIS)

    Self, D.A.; Schroeder, P.C.; Gown, A.M.

    1988-01-01

    Contraction of the follicular wall about the time of ovulation appears to be a coordinated event; however, the cells that mediate it remain poorly studied. We examined the theca externa cells in the wall of hamster follicles for the presence of a functional actomyosin system, both in developing follicles and in culture. We used a monoclonal antibody (HHF35) that recognizes the alpha and gamma isoelectric variants of actin normally found in muscle, but not the beta variant associated with non-muscle sources, to evaluate large preovulatory follicles for actin content and composition. Antibody staining of sectioned ovaries showed intense circumferential reactivity in the outermost wall of developing follicles. Immunoblots from two-dimensional gels of theca externa lysates demonstrated the presence of the two muscle-specific isozymes of actin. Immunofluorescence of cultured follicular cells pulse-labeled with [3H] thymidine (for autoradiographic detection of DNA replication) revealed the presence, in many dividing cells, of actin filaments aligned primarily along the longitudinal axis of the cells. In cultures exposed to the calcium ionophore A23187 (10(-4) M) for varying periods (5 min to 1 h), contraction of many individual muscle-actin-positive cells was observed. Immunofluorescence of these cells, fixed immediately after ionophore-induced contraction, revealed compaction of the actin filaments. Our findings demonstrate that the cells of the theca externa contain muscle actins from an early stage and that these cells are capable of contraction even while proliferating in subconfluent cultures. They suggest that follicular growth may include a naturally occurring developmental sequence in which a contractile cell type proliferates in the differentiated state

  19. Enhanced malignant transformation is accompanied by increased survival recovery after ionizing radiation in Chinese hamster embryo fibroblasts

    International Nuclear Information System (INIS)

    Boothman, D.A.

    1994-01-01

    Transformed Chinese hamster embryo fibroblasts (CHEF), which gradually increase in tumor-forming ability in nude mice, were isolated from normal diploid CHEF/18 cells. Transformed CHEF cells (i.e., T30-4 > 21-2M3 > 21-2 > normal CHEF/18) showed gradual increases in potentially lethal damage (PLD) survival recovery. β-Lapachone and camptothecin, modulators of topoisomerase I (Topo I) activity, not only prevented survival recovery in normal as well as in tumor cells, but enhanced unscheduled DNA synthesis. These seemingly conflicting results are due to the fact that Topo I activity can be modulated by inhibitors to convert single-stranded DNA lesions into double-stranded breaks. Increases in unscheduled DNA synthesis may result from a continual supply of free ends, on which DNA repair processes may act. Altering Topo I activity with modulators appears to increase X-ray lethality via a DNA lesion modification suicide pathway. Cells down-regulate Topo I immediately after ionizing radiation to prevent Topo I-mediated lesion modification and to enhance survival recovery. 16 refs., 3 figs., 1 tab

  20. Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

    International Nuclear Information System (INIS)

    Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A.

    1991-01-01

    1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links

  1. Enhancement of postreplication repair in Chinese hamster cells

    International Nuclear Information System (INIS)

    D'Ambrosio, S.M.; Setlow, R.B.

    1976-01-01

    Alkaline sedimentation profiles of pulse-labeled DNA from Chinese hamster cells showed that DNA from cells treated with N-acetoxy-acetylaminofluorene or ultraviolet radiation was made in segments smaller than those from untreated cells. Cells treated with a small dose (2.5 μM) of N-acetoxy-acetylaminofluorene or(2.5 J . m -2 ) 254-nm radiation, several hours before a larger dose (7 to 10 μM) of N-acetoxy-acetylaminofluorene or 5.0 J . m -2 of 254-nm radiation, also synthesized small DNA after the second dose. However, the rate at which this small DNA was joined together into parental size was appreciably greater than in absence of the small dose. This enhancement of postreplication repair (as a result of the initial small dose) was not observed when cells were incubated with cycloheximide between the two treatments. The results suggest that N-acetoxy-acetylaminofluorene and ultraviolet-damaged DNA from Chinese hamster cells are repaired by similar postreplicative mechanisms that require de novo protein synthesis for enhancement

  2. 12-O-Tetradecanoyl-phorbol-13-acetate and its relationship to SCE induction in Syrian and Chinese hamster cells

    International Nuclear Information System (INIS)

    Popescu, N.C.; Amsbaugh, S.C.; Larramendy, M.L.; DiPaolo

    1982-01-01

    12-O-Tetradecanoyl-phorbol-13-acetate (TPA) in conditions that produce enhancement of ultraviolet light (UV) and x-irradiation Syrian hamster embryo cell (HEC) transformation did not cause further increase in the sister chromatid exchange (SCE) frequency induced by UV and x-irradiation, two physical carcinogens that differ in their mode of DNA interaction and efficiency of SCE induction. Several factors which might influence SCE induction by TPA were studied on HEC and Chinese hamster V79-4 cells. Heat-inactivated serum was used because of the possibility that a serum component may interfere with TPA ability to cause SCE. TPA effect on SCE was determined at the first and second division post treatment on cells exposed to different 5-bromodeoxyuridine (BrdUrd) concentrations. Independent of BrdUrd concentration (1-10μg/ml medium) and the number of cells divisions post treatment, TPA (0.01-2μg/ml medium) was ineffective in inducing SCE in exponentially and stationary HEC cultures cultivated in medium supplemented with heat-inactivated serum. Also, TPA did not increase the SCE frequency in V79-4 Chinese hamster cells cultured in heat-activated or noninactivated serum. Although SCE induction, a cellular response to carcinogen-induced DNA damage, may be important for the induction of transformation by environmental agents, the enhancement of transformation frequency caused by TPA occurs without further DNA alterations involved in SCE formation

  3. Mitotic spindle proteomics in Chinese hamster ovary cells.

    Directory of Open Access Journals (Sweden)

    Mary Kate Bonner

    Full Text Available Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.

  4. Chinese hamster pleiotropic multidrug-resistant cells are not radioresistant

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Gamson, J.; Russo, A.; Friedman, N.; DeGraff, W.; Carmichael, J.; Glatstein, E.

    1988-01-01

    The inherent cellular radiosensitivity of a Chinese hamster ovary pleiotropic cell line that is multidrug resistant (CHRC5) was compared to that of its parental cell line (AuxB1). Radiation survival curve parameters n and D0 were 4.5 and 1.1 Gy, respectively, for the CHRC5 line and 5.0 and 1.2 Gy, respectively, for the parental line. Thus, the inherent radiosensitivity of the two lines was similar even though key intracellular free radical scavenging and detoxifying systems employing glutathione, glutathione transferase, and catalase produced enzyme levels that were 2.0-, 1.9-, and 1.9-fold higher, respectively, in the drug-resistant cell line. Glutathione depletion by buthionine sulfoximine resulted in the same extent of aerobic radiosensitization in both lines (approximately 10%). Incorporation of iododeoxyuridine into cellular DNA sensitized both cell lines to radiation. These studies indicate that pleiotropic drug resistance does not necessarily confer radiation resistance

  5. A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

    KAUST Repository

    Hefzi, Hooman; Ang, Kok  Siong; Hanscho, Michael; Bordbar, Aarash; Ruckerbauer, David; Lakshmanan, Meiyappan; Orellana, Camila  A.; Baycin-Hizal, Deniz; Huang, Yingxiang; Ley, Daniel; Martinez, Veronica  S.; Kyriakopoulos, Sarantos; Jimé nez, Natalia  E.; Zielinski, Daniel  C.; Quek, Lake-Ee; Wulff, Tune; Arnsdorf, Johnny; Li, Shangzhong; Lee, Jae  Seong; Paglia, Giuseppe; Loira, Nicolas; Spahn, Philipp  N.; Pedersen, Lasse  E.; Gutierrez, Jahir  M.; King, Zachary  A.; Lund, Anne  Mathilde; Nagarajan, Harish; Thomas, Alex; Abdel-Haleem, Alyaa M.; Zanghellini, Juergen; Kildegaard, Helene  F.; Voldborg, Bjø rn  G.; Gerdtzen, Ziomara  P.; Betenbaugh, Michael  J.; Palsson, Bernhard  O.; Andersen, Mikael  R.; Nielsen, Lars  K.; Borth, Nicole; Lee, Dong-Yup; Lewis, Nathan  E.

    2016-01-01

    Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess

  6. A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

    DEFF Research Database (Denmark)

    Hefzi, Hooman; Ang, Kok Siong; Hanscho, Michael

    2016-01-01

    Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways...

  7. Suppression of radiation mutagenesis by dactinomycin in Chinese hamster cells

    International Nuclear Information System (INIS)

    Tokita, N.; Capenter, S.G.; Chen, D.J.; MacInnes, M.A.; Raju, M.R.

    1985-01-01

    Dactinomycin (AMD) suppression of radiation mutagenesis was investigated using an in vitro mutation assay (6-thioguanine resistance) in Chinese hamster ovary cells. Cells were exposed to acute single doses of x rays followed by 1 hr-treatment with 0.1 or 1 μg/ml AMD. The cell survival curves plotted as a function of x-ray doses were similar for radiation alone and radiation plus AMD. The results suggest that AMD treatment was only slightly mutagenic, however, when given immediately after irradiation, it suppressed radiatiion mutagenesis at higher x-ray dose regions (below 10% survival levels). Higher AMD concentrations appeared more suppressive than lower concentrations. Dose-response data analyzed based on Poisson distribution models suggest the stochastic dependence of x-ray mutagenesis and AMD cytotoxity

  8. Vitamin K metabolism in Chinese Hamster Ovary cells

    International Nuclear Information System (INIS)

    Hoffman, H.S.

    1986-01-01

    Recent investigations suggest that vitamin K may have functions other than in blood coagulation and calcification. The present study was undertaken to investigate this hypothesis using cells in culture. Chinese Hamster Ovary (CHO) cells were chosen due to their active metabolism and growth and lack of similarity to liver and bone cells, in which vitamin K metabolism is well known. Cells were adapted to serum-free media, incubated in media containing the appropriate concentrations of vitamin K for specified times, scraped from plates, pelleted, extensively washed to remove adhering vitamin K, extracted with chloroform:methanol (2:1, v/v) and analyzed on C18 HPLC columns. Uptake of vitamin K by CHO cells follows saturation kinetics at vitamin K concentrations up to 25 μ M and is transported into cells at the rate of 10 pmol/min. 10 6 cells. After 24 hours, 3 H vitamin K is metabolized by CHO cells to several compounds, the major of which was isolated and identified as vitamin K epoxide. In 3 experiments, after 24 hours, the average cellular uptake of vitamin K was 8% with approximately half being metabolized to vitamin K epoxide. These results demonstrate that vitamin K is metabolized in cells with widely different functions and suggest a generalized function for vitamin K which has yet to be elucidated

  9. Effect of neon ions on synchronized Chinese hamster cells

    International Nuclear Information System (INIS)

    Raju, M.R.; Carpenter, S.G.; Tokita, N.; Howard, J.

    1985-01-01

    The variation in radiosensitivity across the cell cycle after exposure to neon ions and 60 Co γ-rays is reported for cultured hamster cells. The cells were first synchronized by mitotic selection, then resynchronized in the region of the G 1 /S boundary by treatment with 10 -3 M hydroxyurea. Although the use of hydroxyurea improves the synchrony, it does sensitize cells at the G 1 /S boundary to some degree. The cells were exposed at the plateau and the distal peak position of a neon ion beam modified by a 10 cm wide ridge filter. The results indicate that the variation (ratio of maximum to minimum survival after fixed doses of radiation that are approximately matched to produce similar cell killing) was approximately 80 to 100-fold for 60 Co γ-rays and neon ions at the plateau, and 25-fold for distal peak neon ions. While the r.b.e. of distal peak neon ions decreased rapidly with increasing dose for cells in late S-phase, the r.b.e. is independent of dose for cells at the G 1 /S boundary. (author)

  10. Effect of neon ions on synchronized Chinese hamster cells

    Energy Technology Data Exchange (ETDEWEB)

    Raju, M.R.; Carpenter, S.G.; Tokita, N. (Los Alamos National Lab., NM (USA)); Howard, J. (Lawrence Berkeley Lab., CA (USA))

    1985-08-01

    The variation in radiosensitivity across the cell cycle after exposure to neon ions and /sup 60/Co ..gamma..-rays is reported for cultured hamster cells. The cells were first synchronized by mitotic selection, then resynchronized in the region of the G/sub 1//S boundary by treatment with 10/sup -3/ M hydroxyurea. Although the use of hydroxyurea improves the synchrony, it does sensitize cells at the G/sub 1//S boundary to some degree. The cells were exposed at the plateau and the distal peak position of a neon ion beam modified by a 10 cm wide ridge filter. The results indicate that the variation (ratio of maximum to minimum survival after fixed doses of radiation that are approximately matched to produce similar cell killing) was approximately 80 to 100-fold for /sup 60/Co ..gamma..-rays and neon ions at the plateau, and 25-fold for distal peak neon ions. While the r.b.e. of distal peak neon ions decreased rapidly with increasing dose for cells in late S-phase, the r.b.e. is independent of dose for cells at the G/sub 1//S boundary.

  11. Constitutive overexpression of a growth-regulated gene in transformed Chinese hamster and human cells

    International Nuclear Information System (INIS)

    Anisowicz, A.; Bardwell, L.; Sager, R.

    1987-01-01

    Comparison by subtractive hybridization of mRNAs revealed a moderately abundant message in highly tumorigenic CHEF/16 cells present at very low levels in closely related nontumorigenic CHEF/18 cells. After cloning and sequencing the corresponding cDNA, computer comparison showed closest homology with the human connective tissue-activating peptide III (CTAP III). The human tumor cell cDNA hybridizing with the Chinese hamster clone was isolated, sequenced, and found to have closer similarity to the Chinese hamster gene than to CTAP III. Thus, the cloned cDNAs from Chinese hamster and human cells represent a different gene, named gro. Studies of its transcriptional regulation have shown that expression is tightly regulated by growth status in normal Chinese hamster and human cells and relaxed in the tumorigenic cells so far examined

  12. Embryo Cell Membranes Reconstruction by Tensor Voting

    OpenAIRE

    Michelin , Gaël; Guignard , Léo; Fiuza , Ulla-Maj; Malandain , Grégoire

    2014-01-01

    International audience; Image-based studies of developing organs or embryos produce a huge quantity of data. To handle such high-throughput experimental protocols, automated computer-assisted methods are highly desirable. This article aims at designing an efficient cell segmentation method from microscopic images. The proposed approach is twofold: first, cell membranes are enhanced or extracted by the means of structure-based filters, and then perceptual grouping (i.e. tensor voting) allows t...

  13. Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis

    International Nuclear Information System (INIS)

    Bame, K.J.; Kiser, C.S.; Esko, J.D.

    1987-01-01

    The authors have isolated Chinese hamster ovary cell mutants defective in proteoglycan synthesis by radiographic screening for cells unable to incorporate 35 SO 4 into acid-precipitable material. Some mutants did not incorporate 35 SO 4 into acid-precipitable material, whereas others incorporated about 3-fold less radioactivity. HPLC anion exchange chromatographic analysis of radiolabelled glycosaminoglycans isolated from these mutants revealed many are defective in heparan sulfate biosynthesis. Mutants 803 and 677 do not synthesize heparan sulfate, although they produce chondroitin sulfate: strain 803 makes chondroitin sulfate normally, whereas 677 overaccumulates chondroitin sulfate by a factor of three. These mutants fall into the same complementation group, suggesting that the mutations are allelic. A second group of heparan sulfate biosynthetic mutants, consisting of cell lines 625, 668 and 679, produce undersulfated heparan sulfate and normal chondroitin sulfate. Treatment of the chains with nitrous acid should determine the position of the sulfate groups along the chain. These mutants may define a complementation group that is defective in the enzymes which modify the heparan sulfate chain. To increase the authors repertoire of heparan sulfate mutants, they are presently developing an in situ enzyme assay to screen colonies replica plated on filter discs for sulfotransferase defects

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

    Directory of Open Access Journals (Sweden)

    Yamaguchi Ryoji

    2009-10-01

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

  15. Centriole distribution during tripolar mitosis in Chinese hamster ovary cells

    Science.gov (United States)

    1984-01-01

    During bipolar mitosis a pair of centrioles is distributed to each cell but the activities of the two centrioles within the pair are not equivalent. The parent is normally surrounded by a cloud of pericentriolar material that serves as a microtubule-organizing center. The daughter does not become associated with pericentriolar material until it becomes a parent in the next cell cycle (Rieder, C.L., and G. G. Borisy , 1982, Biol. Cell., 44:117-132). We asked whether the microtubule-organizing activity associated with a centriole was dependent on its becoming a parent. We induced multipolar mitosis in Chinese hamster ovary cells by treatment with 0.04 micrograms/ml colcemid for 4 h. After recovery from this colcemid block, the majority of cells divided into two, but 40% divided into three and 2% divided into four. The tripolar mitotic cells were examined by antitubulin immunofluorescence and by high voltage electron microscopy of serial thick (0.25-micron) sections. The electron microscope analysis showed that centriole number was conserved and that the centrioles were distributed among the three spindle poles, generally in a 2:1:1 or 2:2:0 pattern. The first pattern shows that centriole parenting is not prerequisite for association with pole function; the second pattern indicates that centrioles per se are not required at all. However, the frequency of midbody formation and successful division was higher when centrioles were present in the 2:1:1 pattern. We suggest that the centrioles may help the proper distribution and organization of the pericentriolar cloud, which is needed for the formation of a functional spindle pole. PMID:6373793

  16. Cell-cycle distributions and radiation responses of Chinese hamster cells cultured continuously under hypoxic conditions

    International Nuclear Information System (INIS)

    Tokita, N.; Carpenter, S.G.; Raju, M.R.

    1984-01-01

    Cell-cycle distributions were measured by flow cytometry for Chinese hamster (CHO) cells cultured continuously under hypoxic conditions. DNA histograms showed an accumulation of cells in the early S phase followed by a traverse delay through the S phase, and a G 2 block. During hypoxic culturing, cell viability decreased rapidly to less than 0.1% at 120 h. Radiation responses for cells cultured under these conditions showed an extreme radioresistance at 72 h. Results suggest that hypoxia induces a condition similar to cell synchrony which itself changes the radioresistance of hypoxic cells. (author)

  17. DIP and DIP + 2 as glutathione oxidants and radiation sensitizers in cultured Chinese hamster cells

    International Nuclear Information System (INIS)

    Harris, J.W.; Power, J.A.; Kosower, N.S.; Kosower, E.M.

    1975-01-01

    Two diamide analogues, diazene dicarboxylic acid bis (N'-methyl-piperazide) or DIP, and its bis-N'-methyl iodide salt, or DIP + 2, were tested for their ability to penetrate cultured Chinese hamster cells and oxidize intracellular glutathione. DIP penetrated the cells at a reasonable rate at 18 0 C, 160 nmoles being required to oxidize the endogenous glutathione of 2 x 10 6 cells, but it penetrated very slowly at 0 0 C. DIP + 2 did not effectively oxidize glutathione in Chinese hamster cells, possibly because it did not enter the cels. DIP became toxic after about 10 min of exposure, but its toxicity could be moderated by using anoxic conditions. DIP, but not DIP + 2, sensitized anoxic Chinese hamster cells to X-radiation by a factor of 1.5, an effect that was due entirely to removal of the shoulder from the survival curve. (author)

  18. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  19. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

  20. Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

    Directory of Open Access Journals (Sweden)

    Zhao Roong

    2001-12-01

    Full Text Available Abstract Background Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites. Results Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the α-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern. Conclusion Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This

  1. Propagation of avian metapneumovirus subtypes A and B using chicken embryo related and other cell systems.

    Science.gov (United States)

    Coswig, Lia Treptow; dos Santos, Márcia Bianchi; Hafez, Hafez Mohamed; Ferreira, Helena Lage; Arns, Clarice Weis

    2010-07-01

    Primary isolation of avian metapneumovirus (aMPV) is carried out using tracheal organ culture (TOC) or chicken embryonated eggs with subsequent adaptation in chicken embryo fibroblasts (CEF) or Vero cultures. This study was conducted to evaluate six different cell lines and two avian culture systems for the propagation of aMPV subtypes A and B. The chicken embryo related (CER) cells were used successfully for primary isolation. In addition to Vero and baby hamster kidney (BHK-21) cells, CER cells were also shown to be the most appropriate for propagation of aMPV considering high titres. Propagation of A and B subtypes in CEF and TOC remained efficient after the primary isolation and several passages of viruses in the CER cell line. The growth curves were created using CER, Vero and BHK-21 cell lines. Compared with growth, both yielded higher titres in CER cells during the first 30 h after infection, but no significant difference was observed in the results obtained from CER and Vero cells. This data show that CER cells are adequate for aMPV subtypes A and B propagation, giving similar results to Vero cells. Copyright 2010 Elsevier B.V. All rights reserved.

  2. Model-based analysis of N-glycosylation in Chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Krambeck, Frederick J.; Bennun, Sandra V; Andersen, Mikael Rørdam

    2017-01-01

    The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower...

  3. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

    DEFF Research Database (Denmark)

    Lewis, Nathan E; Liu, Xin; Li, Yuxiang

    2013-01-01

    stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages...

  4. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

    DEFF Research Database (Denmark)

    Lewis, Nathan E; Liu, Xin; Li, Yuxiang

    2013-01-01

    Chinese hamster ovary (CHO) cells, first isolated in 1957, are the preferred production host for many therapeutic proteins. Although genetic heterogeneity among CHO cell lines has been well documented, a systematic, nucleotide-resolution characterization of their genotypic differences has been st...

  5. Toward genome-scale models of the Chinese hamster ovary cells: incentives, status and perspectives

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder; Fan, Yuzhou; Weilguny, Dietmar

    2014-01-01

    Bioprocessing of the important Chinese hamster ovary (CHO) cell lines used for the production of biopharmaceuticals stands at the brink of several redefining events. In 2011, the field entered the genomics era, which has accelerated omics-based phenotyping of the cell lines. In this review we...

  6. Spermatogonial multiplication in the Chinese hamster. II. Cell cycle properties of undifferentiated spermatogonia

    NARCIS (Netherlands)

    Lok, D.; Jansen, M. T.; de rooij, D. G.

    1983-01-01

    The cell cycle properties of undifferentiated spermatogonia in the Chinese hamster were analysed by the fraction of labelled mitoses technique (FLM) in whole mounted seminiferous tubules. The minimum cell cycle time (Tc) was found to be c. 90 hr for the As and 87 hr for the Apr and Aal

  7. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    International Nuclear Information System (INIS)

    Lam, C.K.C.

    1982-11-01

    Applying radiation directly on cells, far-uv is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6-TG), ouabain and diptheria toxin (DT). Gold light has no killing and mutagenic effects on CHO (Chinese hamster ovary) cells. Use of filters showed that a small percentage of shorter wavelengths in the far-uv region is responsible for most of the killing and mutagenic effects in the unfiltered broad spectra of black and white light

  8. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    Energy Technology Data Exchange (ETDEWEB)

    Lam, C.K.C.

    1982-11-01

    Applying radiation directly on cells, far-uv is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6-TG), ouabain and diptheria toxin (DT). Gold light has no killing and mutagenic effects on CHO (Chinese hamster ovary) cells. Use of filters showed that a small percentage of shorter wavelengths in the far-uv region is responsible for most of the killing and mutagenic effects in the unfiltered broad spectra of black and white light.

  9. Evidence that cell surface charge reduction modifes capillary red cell velocity-flux relationships in hamster cremaster muscle

    NARCIS (Netherlands)

    Vink, H.; Wieringa, P. A.; Spaan, J. A.

    1995-01-01

    1. From capillary red cell velocity (V)-flux (F) relationships of hamster cremaster muscle a yield velocity (VF = 0) can be derived at which red cell flux is zero. Red cell velocity becomes intermittent and/or red blood cells come to a complete standstill for velocities close to this yield velocity,

  10. From stem cell to embryo without centrioles.

    Science.gov (United States)

    Stevens, Naomi R; Raposo, Alexandre A S F; Basto, Renata; St Johnston, Daniel; Raff, Jordan W

    2007-09-04

    Centrosome asymmetry plays a key role in ensuring the asymmetric division of Drosophila neural stem cells (neuroblasts [NBs]) and male germline stem cells (GSCs) [1-3]. In both cases, one centrosome is anchored close to a specific cortical region during interphase, thus defining the orientation of the spindle during the ensuing mitosis. To test whether asymmetric centrosome behavior is a general feature of stem cells, we have studied female GSCs, which divide asymmetrically, producing another GSC and a cystoblast. The cystoblast then divides and matures into an oocyte, a process in which centrosomes exhibit a series of complex behaviors proposed to play a crucial role in oogenesis [4-6]. We show that the interphase centrosome does not define spindle orientation in female GSCs and that DSas-4 mutant GSCs [7], lacking centrioles and centrosomes, invariably divide asymmetrically to produce cystoblasts that proceed normally through oogenesis-remarkably, oocyte specification, microtubule organization, and mRNA localization are all unperturbed. Mature oocytes can be fertilized, but embryos that cannot support centriole replication arrest very early in development. Thus, centrosomes are dispensable for oogenesis but essential for early embryogenesis. These results reveal that asymmetric centrosome behavior is not an essential feature of stem cell divisions.

  11. Serotonergic modulation of hippocampal pyramidal cells in euthermic, cold-acclimated, and hibernating hamsters

    Science.gov (United States)

    Horrigan, D. J.; Horwitz, B. A.; Horowitz, J. M.

    1997-01-01

    Serotonergic fibers project to the hippocampus, a brain area previously shown to have distinctive changes in electroencephalograph (EEG) activity during entrance into and arousal from hibernation. The EEG activity is generated by pyramidal cells in both hibernating and nonhibernating species. Using the brain slice preparation, we characterized serotonergic responses of these CA1 pyramidal cells in euthermic, cold-acclimated, and hibernating Syrian hamsters. Stimulation of Shaffer-collateral/commissural fibers evoked fast synaptic excitation of CA1 pyramidal cells, a response monitored by recording population spikes (the synchronous generation of action potentials). Neuromodulation by serotonin (5-HT) decreased population spike amplitude by 54% in cold-acclimated animals, 80% in hibernating hamsters, and 63% in euthermic animals. The depression was significantly greater in slices from hibernators than from cold-acclimated animals. In slices from euthermic animals, changes in extracellular K+ concentration between 2.5 and 5.0 mM did not significantly alter serotonergic responses. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin mimicked serotonergic inhibition in euthermic hamsters. Results show that 5-HT is a robust neuromodulator not only in euthermic animals but also in cold-acclimated and hibernating hamsters.

  12. Survival and kinetics of Chinese hamster ovary cell subpopulations induced by Adriamycin and radiation

    International Nuclear Information System (INIS)

    Schneiderman, M.H.

    1979-01-01

    Mitotic selection of Chinese hamster ovary (CHO) cells, at 10 min intervals after the initiation of Adriamycin and/or x-ray treatment was used to measure the kinetics and survival of cells which progressed without delay, the ''refractory'' cells, the cells that reached mitosis only after recovery from the treatment-induced delay, the ''recovered'' cells, and the survival of the cells remaining attached to the flask 5 h after treatment. The cell kinetics were determined from the rate at which cells entered mitosis, and the reproductive integrity from the survival of the selected refractory, recovered and remaining (unselected) cells

  13. The effect of hydroxyurea on synchronized Chinese hamster ovary cells irradiated by ultraviolet light

    International Nuclear Information System (INIS)

    Burg, K.; Collins, A.R.S.; Johnson, R.T.

    1979-01-01

    The effect of hydroxyurea (HU) on cell survival was investigated in Chinese hamster ovary cells after different radiation doses of UV light (254 nm) during the individual phases of the cell cycle. HU inhibits the repair DNA replication by mediation through the DNA precursor pool. These results are supported by the absence of the effect of HU both in the G2 phase possessing high levels of precursors and in supplying the 4 deoxyribonucleosides together with HU after irradiation

  14. Response of maternal immune cells of irradiation of mouse embryos

    International Nuclear Information System (INIS)

    Nicholls, E.M.; Markovic, B.

    1988-01-01

    This work began as an attempt to explain the paradox of pregnancy - the survival and growth of the semi-allogenic embryo in an immunologically hostile environment. In 1982 and 1983 we reported the tracing of quinacrine labelled maternal leukocytes (WBC) in maternal, placental and embryonic mouse tissues by fluorescence microscopy. We found that cells in the placenta phagocytose labelled WBC, so that after 1-2 hours the labelled nuclear DNA is found as brightly fluorescing particles in the cytoplasm of the phagocytes with no evidence of it in the nuclei. Identical cells were observed in slide preparations of embryos which had been carefully separated from their placentas. We also found a small population of intact labelled lymphocytes, clearly maternal in origin, in the embryos. This seems to be another paradox - placental phagocytes are observed to be phagocytosing maternal WBC in the placenta and embryo, but there are also free maternal cells in the placenta and embryo. A theoretical explanation is that maternal lymphocytes alloreactive against the embryo will attempt to react with placental cells and in the process be phagocytosed, while other maternal cells will be able to enter the embryo where they could have a surveillance function, removing dead or mutant embryonic cells. To test this theory a series of experiments were carried out and are reported

  15. Detection of programmed cell death in plant embryos.

    Science.gov (United States)

    Filonova, Lada H; Suárez, María F; Bozhkov, Peter V

    2008-01-01

    Programmed cell death (PCD) is an integral part of embryogenesis. In plant embryos, PCD functions during terminal differentiation and elimination of the temporary organ, suspensor, as well as during establishment of provascular system. Embryo abortion is another example of embryonic PCD activated at pathological situations and in polyembryonic seeds. Recent studies identified the sequence of cytological events leading to cellular self-destruction in plant embryos. As in most if not all the developmental cell deaths in plants, embryonic PCD is hallmarked by autophagic degradation of the cytoplasm and nuclear disassembly that includes breakdown of the nuclear envelope and DNA fragmentation. The optimized setup of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) allows the routine in situ analysis of nuclear DNA fragmentation in plant embryos. This chapter provides step-by-step procedure of how to process embryos for TUNEL and how to combine TUNEL with immunolocalization of the protein of interest.

  16. Spontaneous mutation rate in Chinese hamster cell clones differing in UV-sensitivity

    International Nuclear Information System (INIS)

    Manuilova, E.S.; Bagrova, A.M.; Moskovskij Gosudarstvennyj Univ.

    1983-01-01

    The spontaneous rate of appearance of mutations to 6-mercaptopurine (6 MP) resistence in the cells of CHR2 and CHs2 clones dofferent in sensitivity to lethal and matagenous effect of UV-rays, is investigated. Increased UV-sensitivity of CHs2 clone is caused by the violation of postreplicative DNA reparation. It is established that the purity of spontaneously occuring mutations in both clones turns out to be similar, i.e. (1.5-1.8)x10 -5 for the cell pergeneration. It is shown that the effect of postreplicative DNA reparation in the cells of chinese hamster is not connected with the increase of spontaneous mutation ability. The problem on the possible role of reparation in the mechanism of appearance of spontaneous and induced mutations in the cells of Chinese hamster with increased UV-sensitivity is discussed

  17. High resistance of fibroblasts from Mongolian gerbil embryos to cell killing and chromosome aberrations by X-irradiation

    International Nuclear Information System (INIS)

    Suzuki, F.; Nakao, N.; Nikaido, O.; Kondo, S.

    1992-01-01

    Mongolian gerbil (Meriones unguiculatus) is known to be one of the most radioresistant animal species. In order to determine whether there is any correlation between mortality of mammals exposed to γ- or X-rays and radiation sensitivity of culture cells derived from different mammalian species, we have examined the X-ray survival curves of normal diploid fibroblasts from Mongolian gerbil embryos and compared with those of other cultured embryo cells from various laboratory animals and normal human. There was a big difference in cell survival to X-rays among different mammalian species. The D 0 values of Mongolian gerbil cells ranged from 2.3 to 2.6 Gy which are twice as high as those of human cells. The mean D 0 value of human cells was 1.1 Gy. Mouse, rat, Chinese hamster and Syrian/golden hamster cells showed similar D 0 values ranging from 1.7 to 2.0 Gy. When cells were irradiated with 2 Gy of X-rays, three times longer mitotic delay was observed in human cells than in Mongolian gerbil cells. At this X-ray dose, furthermore, ten times more chromosome aberrations were detected in human cells than in Mongolian gerbil cells, and the frequencies of other rodent cells lay between the values for the two cell strains. These data indicate that the Mongolian gerbil cells are resistant to X-ray-induced cell killing and chromosome aberrations, and that radiation sensitivity of primarily cultured mammalian cells may be reflected by their radioresistance in vivo. (author)

  18. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    Science.gov (United States)

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  19. Nuclear scaffold organization in the X-ray sensitive Chinese hamster mutant cell line, xrs-5

    International Nuclear Information System (INIS)

    Yasui, L.S.; Fink, T.J.; Enrique, A.M.

    1994-01-01

    Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. (author)

  20. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    International Nuclear Information System (INIS)

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-01-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts

  1. Analysis of trace elements in chicken embryo cells

    International Nuclear Information System (INIS)

    Qiu Zhijun; Wang Jiqing; Guo Panlin; Li Xiaolin; Zhu Jieqing; Lu Rongrong

    2002-01-01

    A scanning proton microprobe (SPM) with high resolution and high sensitivity was applied to analyze trace elements in chicken embryo forebrain neutron cell and skeletal muscle myotube cell. The absorption of the two different cells to zinc ions, correlation of elements and trace elemental distributions in the cells were studied. The results indicate that the absorptive capacity of the chicken embryo forebrain neuron cell to zinc ions is larger than that of the chicken embryo skeletal muscle myotube cell, and the concentrations of intracellular trace elements such as Cr, Fe, Ni are explicitly higher. The correlations of elements such as S and Zn or Fe and Zn are positive, but the correlations of P and Ni or Cr and Fe are negative. From the maps of cellular elemental distribution the contents of the different elements are different in the intracellular parts, for example, the contents of the elements phosphorus, sulfur, potassium in the cell membranes are higher than that in the cells

  2. Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

    International Nuclear Information System (INIS)

    Rutten, A.A.; Beems, R.B.; Wilmer, J.W.; Feron, V.J.

    1988-01-01

    The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-3H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 micron, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl- 3 H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration

  3. Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids

    International Nuclear Information System (INIS)

    Nairn, R.S.; Humphrey, R.M.; Adair, G.M.

    1988-01-01

    Transfection of UV-hypersensitive, DNA repair-deficient Chinese hamster ovary (CHO) cell lines and parental, repair-proficient CHO cells with UV-irradiated pHaprt-1 or pSV2gpt plasmids resulted in different responses by recipient cell lines to UV damage in transfected DNA. Unlike results reported for human cells, UV irradiation of transfecting DNA did not stimulate genetic transformation of CHO recipient cells. In repair-deficient CHO cells, proportionally fewer transformants were produced with increasing UV damage than in repair-proficient cells in transfections with UV-irradiated hamster adenine phosphoribosyltransferase (APRT) gene contained in plasmid pHaprt-1. Transfection of CHO cells with UV-irradiated pSV2gpt resulted in neither decline in transformation frequencies in repair-deficient cell lines relative to repair-proficient cells nor stimulation of genetic transformation by UV damage in the plasmid. Blot hybridization analysis of DNA samples isolated from transformed cells showed no dramatic changes in copy number or arrangement of transfected plasmid DNA with increasing UV dose. The authors conclude responses of recipient cells to UV-damaged transfecting plasmids depend on type of recipient cell and characteristics of the genetic sequence used for transfection. (author)

  4. Interspecies complementation analysis of xeroderma pigmentosum and UV-sensitive Chinese hamster cells

    International Nuclear Information System (INIS)

    Stefanini, M.; Keijzer, W.; Westerveld, A.; Bootsma, D.

    1985-01-01

    Complementation analysis was performed 24 h after fusion of UV-sensitive CHO cells (CHO 12 RO) with XP cells of complementation groups A, B, C, D, F and G. The parental cells are characterized by low levels of unscheduled DNA synthesis (UDS). In all combinations, the UDS levels observed in heterokaryons were higher than those in parental mutant cells, clearly indicating cooperation of human and Chinese hamster repair functions. In heterokaryons of CHO 12 RO with XP-A and XP-C cells, the UDS values reached about the normal human level, whereas in heterokaryons with XP-B, XP-D and XP-F, UDS was restored at a level approaching that in wild-type CHO cells. The results obtained after fusion of CHO cells with two representative cell strains from the XP-G group, XP 2 BI and XP 3 BR, were inconsistent. Fusion with XP 3 BR cells yielded UDS levels ranging from wild-type Chinese hamster to normal human, whereas fusion with XP 2 BI cells resulted in a slight increase in UDS which even after 48 h remained below the level found in wild-type CHO cells. The occurrence of complementation in these interspecies heterokaryons indicates that the genetic defect in the CHO 12 RO cells is different from the defects in the XP complementation groups tested

  5. Comparative ecotoxicity of potential biofuels to water flea (Daphnia magna), zebrafish (Danio rerio) and Chinese hamster (Cricetulus griseus) V79 cells.

    Science.gov (United States)

    Heger, Sebastian; Du, Miaomiao; Bauer, Kevin; Schäffer, Andreas; Hollert, Henner

    2018-08-01

    The ecotoxicity of two biofuel candidates (1‑octanol and 2‑butanone) was investigated by an integrative test strategy using three bioassays: the acute immobilisation test with water flea (D. magna), the fish embryo acute toxicity test with zebrafish (Danio rerio) and the in vitro micronucleus assay with Chinese hamster (Cricetulus griseus) V79 cells. The median effective concentration (EC 50 ) values were 14.9±0.66mgL -1 for 1‑octanol, and 2152.1±44.6mgL -1 for 2‑butanone in the D. magna test. Both 1‑octanol and 2‑butanone caused teratogenic and lethal effects on zebrafish embryos, while exposure to 1‑octanol significantly induced these effects at concentrations ≥2.0mgL -1 . These results indicate that 1‑octanol exert much higher ecotoxicity than 2‑butanone to D. magna and zebrafish embryos. Moreover, both 1‑octanol and 2‑butanone did not cause significant genotoxic effects, while their metabolites significantly induced micronuclei in V79 cells. The present study proposed an integrative test approach to evaluate the potential ecotoxicity of biofuels using simple, quick and inexpensive bioassays. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

    International Nuclear Information System (INIS)

    Stamato, T.D.; Weinstein, R.; Giaccia, A.; Mackenzie, L.

    1983-01-01

    A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage

  7. Culture conditions affecting the survival response of Chinese hamster ovary cells treated by hyperthermia

    International Nuclear Information System (INIS)

    Highfield, D.P.; Holahan, E.V.; Dewey, W.C.

    1982-01-01

    Using lethally irradiated feeder cells to control cell population densities, researchers investigated the survival of Chinese hamster ovary cells heated between 42.2 and 45.5 degrees C. Test cells were plated into T25 flasks with or without feeder cells, incubated 2 hours at 37 degrees C, and then given various heat treatments. Under all heating conditions, survival increased in those flasks containing feeder cells. Increased survival (by as much as a factor of 100 for cells heated at 42.4 degrees C for 6-10 hr) was most apparent when cells were heated to thermotolerance. By adjustment of test and feeder cell numbers, survival increased as density increased; however, maximum survival followed a transition period that occurred between the plating of 1 X 10(4) and 6 X 10(4) cells. Experimental artifacts due to improper control of cell density was demonstrated

  8. Recovery from DNA synthesis in V 79 chinese hamster cells irradiated with UV light

    International Nuclear Information System (INIS)

    Ventura, A.M.

    1987-01-01

    Mammalian cells recover from DNA synthesis inhibition by UV light before most of the pyrimidine dimers have been removed from the genome. Most of the rodent cells show a deficient dimer excision repair compared with normal human fibroblasts. Despite this fact they recover efficiently from DNA synthesis inhibition after UV. In Chinese hamster V 79 cells was found that this recovery takes place in the absence of a significant excision repair, and it seems to be directly coupled to a recovery in the rate of movement of the replication fork. 120 refs, 31 figs. (author)

  9. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.

    OpenAIRE

    Kaina, B; Lohrer, H; Karin, M; Herrlich, P

    1990-01-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of re...

  10. Rate of oxygen consumption of hamster melanoma cells as a factor influencing their radioresistance

    International Nuclear Information System (INIS)

    Pajak, S.; Subczynski, W.; Panz, T.; Lukiewicz, S.

    1980-01-01

    It has been reported in recent years that the level of radiosensitivity of neoplasmic cells in vivo and of sphaeroids in vitro can be modified by controlling their rate of oxygen consumption. Thus, an attempt was made to compare this rate in the case of the melanotic and amelanotic lines of Bomirski hamster melanoma in vitro, as it is known that these two lines distinctly differ in their reactivity to ionizing radiations. The measurements carried out by the use of a new ESR method revealed that pigmented and pigmentless cells consume oxygen at significantly different rates. This means that oxygen utilization may contribute to the overall level of radioresistance of melanoma cells. (author)

  11. Effects of insulin on the survival of irradiated chinese hamster lung cells

    Energy Technology Data Exchange (ETDEWEB)

    Lin, P S; Kwock, L; Hefter, K; Wallach, D F.H.; Brotman, R [Tufts-New England Medical Center, Boston, Mass. (USA)

    1977-01-01

    Insulin treatment (10/sup -7/-10/sup -9/ M) before ..gamma.. irradiation (50 to 500 rads) increases the long term survival of Chinese hamster lung cells (DON). Our data indicates that the radioprotective effect of insulin is not due to a modulation of cyclic-adenosine-3',5'-monophosphate levels within these cells. The results suggest that the radiosensitive plasma membrane component postulated to be involved in the interphase death of thymocytes and protected by insulin may have a counterpart in DON cells.

  12. Cell killing and mutation induction on Chinese hamster cells by photoradiations

    International Nuclear Information System (INIS)

    Lam, C.K.C.

    1982-01-01

    The subject matter of this investigation concerns the killing and mutagenic effects induced by far-UV radiation and broad spectra of black, white and gold lights. Applying radiation directly on CHO (Chinese hamster ovary) cells, far-UV is more effective than black light, and black light is more effective than white light in inducing proliferative death and in inducing resistance to 6-thioguanine (6TG), ouabain and diptheria toxin (DT). Cells in the G1/early S boundary are the most sensitive to far-UV or unfiltered fluorescent lights. When synchronous cells are irradiated with moderate doses of far-UV or unfiltered broad spectra of black light, mutations to 6-TG and ouabain resistance are slightly higher in early S period than in the remaining parts of the cell cycle. Mutation induction of 6-TG, ouabain or DT resistance is increased in the split-dose samples of the asynchronous and synchronous CHO cells. CHO cells predominantly express an error-prone repair mechanism after photoirradiation

  13. CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

    Science.gov (United States)

    Yong, Yu-Le; Liao, Cheng-Gong; Wei, Ding; Chen, Zhi-Nan; Bian, Huijie

    2016-04-01

    CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells. © 2015 International Federation for Cell Biology.

  14. Postreplication gap filling in the DNA of X-ray-damaged Chinese hamster cells

    International Nuclear Information System (INIS)

    Koerner, I.; Malz, W.

    1975-01-01

    In X-irradiated Chinese hamster cells the newly synthesized DNA has a lower molecular weight than the DNA in control cells. This reduced molecular weight has been interpreted by gap induction opposite the lesions in the parental DNA strands. Within two hours these postreplication gaps were closed. With the aid of BrdUrd-photolys-technique it could be demonstrated that the gaps were filled by de novo synthesis. But we were not able to show a participation of parental DNA in the gap-filling process

  15. Variations in sensitivity of synchronized Chinese hamster cells to oxic and anoxic X-ray exposures

    International Nuclear Information System (INIS)

    Siracka, E.; Littbrand, B.; Clifton, K.H.; Revesz, L.

    1975-01-01

    V-79 Chinese hamster cells in monolayer cultures on glass surfaces were synchronized by treatment with hydroxyurea and then exposed at different times to X-rays in air or in oxygen-free argon. Survival determinations indicated that the oxygen enhancement ratio (OER) as expressed by the ratio of the respective D 0 values varied over a narrow range in the different phases of the cell cycle. These changes resulted from cyclic alterations in both aerobic and anaerobic D 0 values, possibly in n values. (author)

  16. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

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

    1986-01-01

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs

  17. [Specification of cell destiny in early Caenorhabditis elegans embryo].

    Science.gov (United States)

    Schierenberg, E

    1997-02-01

    Embryogenesis of the nematode Caenorhabditis elegans has been described completely on a cell-by-cell basis and found to be essentially invariant. With this knowledge in hands, micromanipulated embryos and mutants have been analyzed for cell lineage defects and the distribution of specific gene products. The results challenge the classical view of cell-autonomous development in nematodes and indicate that the early embryo of C. elegans is a highly dynamic system. A network of inductive events between neighboring cells is being revealed, which is necessary to assign different developmental programs to blastomeres. In those cases where molecules involved in these cell-cell interactions have been identified, homologies to cell surface receptors, ligands and transcription factors found in other systems have become obvious.

  18. Isolation and characterization of variant clones of Chinese hamster cells after treatment with irradiated 5-iodouridine

    International Nuclear Information System (INIS)

    Kuroda, Y.; Yokoiyama, A.; Kada, T.

    1975-01-01

    Variant clones were isolated from cultured Chinese hamster Don cells after treatment with irradiated 5-iodouridine. The following characters of a primary variant clone, C-11 and a secondary variant clone, C-24 were compared with those of the original clone C-1: colony-forming activity, growth rate in the presence of irradiated and unirradiated 5-iodouridine, distribution of chromosome numbers and cell cohesion. The variant clones C-11 and C-24 were partially resistant to unirradiated 5-iodouridine at lower concentration and C-24 cells were slightly resistant to short-term treatment with irradiated 5-iodouridine. Unlike clones C-1 and C-11, the variant clone C-24 showed no lag phase on growth in 5-iodouridine medium. The modal numbers of the chromosomes of all three clones were 22, like that of normal Chinese hamster diploid cells. Of the three clones, the variant C-24 cells showed the least mutual cohesion and the original C-1 cells showed the most. The possibility that an alteration in cellular membrane might be related to an increase in the resistance to radiosensitizing agents was discussed

  19. Differential display technique of RNA from tumorigenic and non-tumorigenic variants of hamster cells transformed with avian sarcoma virus

    International Nuclear Information System (INIS)

    Leksa, V.; Altaner, C.

    1997-01-01

    Differential display technique was applied to study expression of RNA in tumorigenic and non-tumorigenic cell variants of avian sarcoma virus transformed hamster cells. Methodical conditions were worked out, which allowed identifying a cDNA fragment of an unknown gene expressed in non-tumorigenic cell variant only. Its role in tumor suppression remains to be determined. (author)

  20. Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

    DEFF Research Database (Denmark)

    Ley, Daniel; Kazemi Seresht, Ali; Engmark, Mikael

    2015-01-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied...

  1. Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

    DEFF Research Database (Denmark)

    Hansen, Henning Gram; Pristovsek, Nusa; Kildegaard, Helene Faustrup

    2017-01-01

    Chinese hamster ovary (CHO) cells are the preferred cell factory for the production of therapeutic glycoproteins. Although efforts primarily within bioprocess optimization have led to increased product titers of recombinant proteins (r-proteins) expressed in CHO cells, post-transcriptional bottle...

  2. Effects of ultrasound, cysteamine, and x-rays on V79 Chinese hamster cells

    International Nuclear Information System (INIS)

    Fu, Y.K.

    1978-01-01

    Chinese hamster V79 cells were exposed to different intensities and durations of 1 MHz ultrasound and were studied for cell lysis and colony-forming ability. The threshold for cell lysis and loss of viability (reduction in colony-forming ability) were found to be ultrasonic intensity, sonication duration, and energy dependent. Above the threshold there was an initial correlation between ultrasound intensity and biological effect: the higher the intensity, the greater the amount of cell lysis and reduction in colony-forming ability. These effects were maximal at an intensity of 10-20 W/cm 2 , and were less at 30 W/cm 2 . Cells were also exposed to ultrasound in the presence of cysteamine and results are compared with the effects of x radiation on cells exposed in the presence of cysteamine or methionine

  3. Studies on adaptive responses in Chinese hamster cells

    International Nuclear Information System (INIS)

    Michelin, S.C.; Perez, M.R. Del; Dubner, D.; Gisone, P.A.

    1997-01-01

    For many years the possibility has been considered of low doses of radiation inducing adaptive responses in cells and organisms against the mutagenic effects of radiation. Currently, a number of experimental data appraise the existence of an adaptive response that is characterized by a decrease of radiation induced genetic damages. The understanding of the molecular mechanism involved in this phenomenon permits to estimate the effects and risks of low dose exposure. In this work, preliminary results of studies on the induction of adaptive response in cells subjected to different doses of ionizing radiation are presented

  4. DNA repair in human xeroderma pigmentosum and Chinese hamster cells

    NARCIS (Netherlands)

    B. Zelle (Bauke)

    1980-01-01

    textabstractAn important feature of living cells is their capacity to maintain the integrity of their hereditary material, the DNA. DNA can be damaged by a variety of physical and chemical agents, among which ultraviolet radiation (UV), ion1z1ng radiation and chemical carcinogens as

  5. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Soares, Carlos Roberto Jorge

    2000-01-01

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 μg hPRU10 6 cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 μg hPRU10 6 cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a reversed

  6. Interchromosomal distribution of gamma ray-induced chromatid aberrations in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Martinez-Lopez, Wilner; Porro, Valentina; Folle, Gustavo A.; Mendez-Acuna, Leticia; Obe, Guenter; Savage, John R.K.

    2000-01-01

    Inter chromosomal distributions of breakpoints from chromatid-type aberrations induced by gamma rays in Chinese hamster ovary cells were analyzed. In most chromosomes the distribution was as expected from chromosome lengths for simple breaks or the respective relative corrected length in case of exchanges. There were deviations from expectation in a few chromosomes for chromatid breaks, interchanges, intra-arm intra changes and inter-arm intra changes. Especially interesting are the results concerning chromosomes 2 and 8, which were more often involved in exchanges than expected. An 'exchange phenotype' for these chromosomes is proposed and possible explanations for the nonrandom distribution of chromosome breakpoints are presented. (author)

  7. Effect of caffeine on the ultraviolet light induction of SV40 virus from transformed hamster cells

    International Nuclear Information System (INIS)

    Zamansky, G.B.; Kleinman, L.F.; Little, J.B.; Black, P.H.; Kaplan, J.C.

    1976-01-01

    The effect of caffeine on the uv light induction of SV40 virus from two transformed hamster cell lines heterogeneous for the induction of infectious virus was studied. The amount of virus induced was significantly increased in both cell lines when exposure to uv light was followed by treatment with caffeine. Caffeine in the absence of uv irradiation did not stimulate virus induction, nor did it stimulate SV40 replication in a lytic infection. There was an apparent difference in the concentrations of caffeine which maximally stimulated SV40 virus induction in the two cell lines. This effect could not be explained by differences in cell survival after exposure to uv light and caffeine. Since caffeine is known to cause the accumulation of gaps formed in DNA during postreplication repair of uv-irradiated rodent cells, our results support the hypothesis that the formation of gaps or breaks in DNA is an important early step in virus induction

  8. Biophysical interpretation of the response of Chinese hamster cells to 24 keV neutrons

    International Nuclear Information System (INIS)

    Holt, P.D.

    1988-01-01

    The response of V79 Chinese hamster cells to a 24 keV neutron spectrum has been compared with data for the response of V79 cells to a range of higher neutron energies (up to 15 MeV). The linear energy transfer (LET) distributions of the neutron spectra were calculated and the expected responses of the cells to the different spectra were calculated using published track-segment data on the response of V79 cells to charged particles with various LET values. The response of the cells to 24 keV neutrons was predicted satisfactorily by the LET distribution, in spite of the fact that the maximum range of the recoil protons is only 0.5 μm. The response was not correctly predicted by the microdosimetric parameter y-bar D * evaluated in a 1 μm diameter sphere. (author)

  9. Telomere Length Reprogramming in Embryos and Stem Cells

    Directory of Open Access Journals (Sweden)

    Keri Kalmbach

    2014-01-01

    Full Text Available Telomeres protect and cap linear chromosome ends, yet these genomic buffers erode over an organism’s lifespan. Short telomeres have been associated with many age-related conditions in humans, and genetic mutations resulting in short telomeres in humans manifest as syndromes of precocious aging. In women, telomere length limits a fertilized egg’s capacity to develop into a healthy embryo. Thus, telomere length must be reset with each subsequent generation. Although telomerase is purportedly responsible for restoring telomere DNA, recent studies have elucidated the role of alternative telomeres lengthening mechanisms in the reprogramming of early embryos and stem cells, which we review here.

  10. Rayleigh instability of the inverted one-cell amphibian embryo

    International Nuclear Information System (INIS)

    Nouri, Comron; Gordon, Richard; Luppes, Roel; Veldman, Arthur E P; Tuszynski, Jack A

    2008-01-01

    The one-cell amphibian embryo is modeled as a rigid spherical shell containing equal volumes of two immiscible fluids with different densities and viscosities and a surface tension between them. The fluids represent denser yolk in the bottom hemisphere and clearer cytoplasm and the germinal vesicle in the top hemisphere. The unstable equilibrium configuration of the inverted system (the heavier fluid on top) depends on the value of the contact angle. The theoretically calculated normal modes of perturbation and the instability of each mode are in agreement with the results from ComFlo computational fluid dynamic simulations of the same system. The two dominant types of modes of perturbation give rise to axisymmetric and asymmetric sloshing of the cytoplasm of the inverted embryos, respectively. This work quantifies our hypothesis that the axisymmetric mode corresponds to failure of development, and the asymmetric sloshing mode corresponds to development proceeding normally, but with reversed pigmentation, for inverted embryos

  11. Modification of potentially lethal damage in irradiated Chinese hamster V79 cells after incorporation of halogenated pyrimidines

    NARCIS (Netherlands)

    Franken, N. A.; van Bree, C. V.; Kipp, J. B.; Barendsen, G. W.

    1997-01-01

    Radiosensitization of exponentially growing and plateau phase Chinese hamster V79 cells by incorporation of halogenated pyrimidines (HP) was investigated for different culture conditions that influenced repair. For this purpose cells were grown for 72 h with 0, 1, 2 and 4 microM of chloro-(CldUrd),

  12. Interphase death of dividing cells. Kinetics of death of cultured Chinese hamster fibroblasts after irradiation with various doses

    International Nuclear Information System (INIS)

    Kublik, L.N.; Veksler, A.M.; Ehjdus, L.Kh.

    1989-01-01

    In studying the kinetics of interphase death (ID) of cultured Chinese hamster cells after irradiation with doses of 100 to 800 Gy the authors showed an increase in the ID rate with increasing radiation dose; the presence of serum in the medium both during and after irradiation prevents the cell death

  13. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most....... Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance...... property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production....

  14. Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

    Science.gov (United States)

    Balbach, Sebastian T; Boiani, Michele

    2015-01-01

    Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

  15. Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

    International Nuclear Information System (INIS)

    Olive, P.L.; Vanderbyl, S.; MacPhail, S.H.

    1991-01-01

    Exponentially growing Chinese hamster V79-171b lung fibroblasts seeded at high density on plastic (approximately 7 x 10(3) cells/cm2) flatten, elongate, and produce significant amounts of extracellular fibronectin. When lysed in weak alkali/high salt, the rate of DNA denaturation following exposure to ionizing radiation is exponential. Conversely, cells plated at low density (approximately 7 x 10(2) cells/cm2) on plastic are more rounded 24 h later, produce little extracellular fibronectin, and display unusual DNA denaturation kinetics after X-irradiation. DNA in these cells resists denaturation, as though constraints to DNA unwinding have developed. Cell doubling time and distribution of cells in the growth cycle are identical for both high and low density cultures as is cell survival in response to radiation damage. The connection between DNA conformation and cell shape was examined further in low density cultures grown in conditioned medium. Under these conditions, cells at low density were able to elongate, and DNA denaturation of low density cultures was identical to that of high density cultures. Conversely, cytochalasin D, which interferes with actin polymerization causing cells to round up and release fibronectin, allowed development of constraints in high density cultures. These results suggest that DNA conformation is sensitive to changes in cell shape which result when cells are grown in different environments. However, these changes in DNA conformation detected by the DNA unwinding assay do not appear to play a direct role in radiation-induced cell killing

  16. N-ethylmaleimide sensitization of x-irradiated hypoxic Chinese hamster cells

    International Nuclear Information System (INIS)

    Kimler, B.F.; Sinclair, W.K.; Elkind, M.M.

    1977-01-01

    Chinese hamster cells were x irradiated either aerobically or hypoxically, after flushing with nitrogen plus carbon dioxide. In agreement with earlier data, for asynchronous cells, the oxygen enhancement ratio (OER) was approximately three. If the sulfhydryl-binding agent N-ethylmaleimide (NEM) was present during or immediately after irradiation, the principal effect was a pronounced decrease in the extrapolation number of the survival curve of NEM-treated cells compared to nontreated cells. This was observed with hypoxic as well as aerobic cells and the OER for NEM-treated cells was also about three. For NEM treatments which were essentially nontoxic, NEM acts synergistically with X rays, suggestive of an inhibition by NEM of a cell's ability to repair sublethal damage. For synchronous cells obtained by mitotic selection, a result consistent with the above was obtained; a dose three times as large was necessary to reduce survival to the same level for hypoxic cells as for aerobic cells, whether or not the cells were treated with NEM. Thus the OER was independent of NEM treatment throughout the cell cycle, with the possible exception of mitosis which could not be studied with the methods used. It is concluded that the action of NEM at low concentrations (0.75 μM) is largely independent of oxygen tension. Oxygen acts to produce more damage per unit dose in the cell while NEM sensitizes apparently by preventing the repair of sublethal damage

  17. Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

    Directory of Open Access Journals (Sweden)

    Zeinab Soleimanifar

    2016-10-01

    Full Text Available Background: Luteinizing hormone (LH was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly.

  18. Effects of 60Co gamma-rays on some biological characteristics of Chinese hamster lung cells

    International Nuclear Information System (INIS)

    Tang Pei; Wang Shoufang; Zhang Shuxian

    1988-01-01

    The proliferation of cells and the relationship between survival and dose were investigated in Chinese hamster lung (CHL) cells grown at stationary phase and irradiated with 60 Co gamma-rays. The ultrastructural changes and chromosome aberration in the cells after irradiation were also observed. The frequency of chromosome aberrations increased linearly with dose and the yields of dicentrics plus rings were best fitted to a linear-quadratic model. The 50% growth-inhibited dose was found to be 4.0Gy. Electron microscopy observation revealed swelling and vacuolation of mitochodria and indistinct cristae at lower doses. The alterations in nucleus at higher doses appeared to be depression of nuclear membrane and disappearance of chromatin

  19. Embryo quality predictive models based on cumulus cells gene expression

    Directory of Open Access Journals (Sweden)

    Devjak R

    2016-06-01

    Full Text Available Since the introduction of in vitro fertilization (IVF in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.

  20. Effects of proliferation on the decay of thermotolerance in Chinese hamster cells.

    Science.gov (United States)

    Armour, E P; Li, G C; Hahn, G M

    1985-09-01

    Development and decay of thermotolerance were observed in Chinese hamster HA-1 cells. The thermotolerance kinetics of exponentially growing and fed plateau-phase cells were compared. Following a 10-min heat exposure at 45 degrees C, cells in both growth states had similar rates of development of tolerance to a subsequent 45-min exposure at 45 degrees C. This thermotolerant state started to decay between 12 and 24 hr after the initial heat exposure. The decay appeared to initiate slightly sooner in the exponentially growing cells when compared to the fed plateau-phase cells. During the decay phase, the rate of thermotolerance decay was similar in the two growth conditions. In other experiments, cells were induced to divide at a slower rate by chronic growth (3 months) in a low concentration of fetal calf serum. Under these low serum conditions cells became more sensitive to heat and the rate of decay of thermotolerance remained the same for exponentially growing cells. Plateau-phase cells were also more sensitive, but thermotolerance decayed more rapidly in these cells. Although dramatic cell cycle perturbations were seen in the exponentially growing cells, these changes appeared not to be related to thermotolerance kinetics.

  1. Use of "excess" human embryos for stem cell research: protecting women's rights and health.

    Science.gov (United States)

    Cohen, C B

    2000-01-01

    Proposed National Institutes of Health guidelines for stem cell research are too narrowly drawn and do not adequately protect the freedom of choice and health of women who donate embryos. They need to be expanded to cover not only the point of embryo donation, but also that of embryo creation. Guidelines are provided to ensure that donors undergoing hyperstimulation and egg retrieval gave voluntary informed consent to the production of embryos that might later prove in excess. A standard for determining when embryos have been overproduced is presented to address the possibility that additional embryos will be created for stem cell research in violation of the guidelines and at risk to women's health.

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

    Science.gov (United States)

    Toyohara, Yukiyo; Hashitani, Susumu; Kishimoto, Hiromitsu; Noguchi, Kazuma; Yamamoto, Nobuto; Urade, Masahiro

    2011-07-01

    This study investigated the inhibitory effect of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) on carcinogenesis and tumor growth, using a 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced hamster cheek pouch carcinogenesis model, as well as the cytocidal effect of activated macrophages against HCPC-1, a cell line established from DMBA-induced cheek pouch carcinoma. DMBA application induced squamous cell carcinoma in all 15 hamsters of the control group at approximately 10 weeks, and all 15 hamsters died of tumor burden within 20 weeks. By contrast, 2 out of the 14 hamsters with GcMAF administration did not develop tumors and the remaining 12 hamsters showed a significant delay of tumor development for approximately 3.5 weeks. The growth of tumors formed was significantly suppressed and none of the hamsters died within the 20 weeks during which they were observed. When GcMAF administration was stopped at the 13th week of the experiment in 4 out of the 14 hamsters in the GcMAF-treated group, tumor growth was promoted, but none of the mice died within the 20-week period. On the other hand, when GcMAF administration was commenced after the 13th week in 5 out of the 15 hamsters in the control group, tumor growth was slightly suppressed and all 15 hamsters died of tumor burden. However, the mean survival time was significantly extended. GcMAF treatment activated peritoneal macrophages in vitro and in vivo, and these activated macrophages exhibited a marked cytocidal effect on HCPC-1 cells. Furthermore, the cytocidal effect of activated macrophages was enhanced by the addition of tumor-bearing hamster serum. These findings indicated that GcMAF possesses an inhibitory effect on tumor development and growth in a DMBA-induced hamster cheek pouch carcinogenesis model.

  3. Recovery of subchromosomal DNA synthesis in synchronous V-79 Chinese hamster cells after ultraviolet light exposure

    International Nuclear Information System (INIS)

    Meechan, P.J.; Carpenter, J.G.

    1986-01-01

    Previous work obtained from Chinese hamster V-79 cells indicated that, immediately following exposure, UV-induced lesions acted as blocks to elongation of nascent strands, but gradually lost that ability over a 10 h period after exposure to 10 J/m 2 . The work reported herein attempted to examine possible cell cycle mediated alterations in the recovery of DNA synthesis. Kinetic incorporation of radiolabeled thymidine studies indicated that there may have been a more rapid recover of DNA synthesis in cells irradiated in G 1 or G 2 vs cells irradiated in S phase. DNA fiber autoradiograms prepared from synchronous cells indicated that after irradiation in any phase of the cell cycle, the length of newly synthesized DNA was equal to control lengths 1 h after exposure to 5.0Jm 2 (or 1 h after entering S phase for cells irradiated in G 1 or G 2 ). This observed recovery was not solely due to an excision process. No cell cycle mediated difference in the number of dimers induced or removed as a function of cell cycle position was observed. These results appear to be consistent with a continuum of effects, with initiation effects dominating the response at low fluences, gapped synthesis at intermediate fluences and elongation inhibition at high fluences. The fluences at which each event dominates may be cell-line specific. (author)

  4. Caffeine-enhanced survival of radiation-sensitive, repair-deficient Chinese hamster cells

    International Nuclear Information System (INIS)

    Utsumi, H.; Elkind, M.M.

    1983-01-01

    A clone of V79 Chinese hamster cells (V79-AL162/S-10) with unique properties has been isolated after a challenge of parental cells (V79-AL162) with 1 mM ouabain. Compared with parental cells, or with other clones isolated after the ouabain challenge, these cells form smaller colonies, are more sensitive to both x rays and fission-spectrum neutrons, and respond atypically to a postirradiation treatment with caffeine. Their enhanced response to x rays results mainly from a large reduction in the shoulder of their survival curve, probably because in late S phase, the most resistant phase in the cell cycle, the survival curve of these cells has a reduced shoulder width. Caffeine, and to a lesser extent theophylline, added to the colony-forming medium immediately after exposure appreciably increases the width of the shoulder of these sensitive cells, whereas caffeine has the opposite effect on the response of normal V79 cells. Thus the unique response of the V79-AL162/S-10 cells to a radiation posttreatment with caffeine (increased survival) results from a net increase in their ability to repair damage that is otherwise lethal; caffeine treatment ordinarly prevents normal V79 cells from repairing damage that is only potentially lethal

  5. Expression of a human gene for polyamine transport in Chinese-hamster ovary cells.

    Science.gov (United States)

    Byers, T L; Wechter, R; Nuttall, M E; Pegg, A E

    1989-01-01

    A molecular-genetic approach towards isolating mammalian polyamine-transport genes and their encoded proteins was devised involving the production of Chinese-hamster ovary (CHO) cells expressing a human polyamine-transport protein. CHO cells and a polyamine-transport-deficient CHO mutant cell line (CHOMG) were equally sensitive to the antiproliferative effects of alpha-difluoromethylornithine (DFMO), which blocked endogenous polyamine synthesis. Exposure to exogenous polyamines increased intracellular polyamine levels and reversed this DFMO-induced cytostasis in the CHO cells, but not in the CHOMG cells. CHOMG cells were therefore transfected with human DNA (isolated from HT-29 colon carcinoma cells) and cells expressing the human polyamine-transport system were identified by the ability of these cells to grow in a medium containing DFMO and polyamines. A number of different positive clones were identified and shown to have the capacity for polyamine uptake and an increased sensitivity to the toxic effects of the polyamine analogue methylglyoxal bis(guanylhydrazone). Differences in these properties between the clones are consistent with a multiplicity of polyamine-transport systems. Some clones also showed a change in growth characteristics, which may indicate a relationship between genes involved in the polyamine-transport system and in cell proliferation. PMID:2512913

  6. Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in Chinese hamster V79 cells by tritium suicide

    International Nuclear Information System (INIS)

    Bryant, R.E.; Schauer, I.E.; Hatcher, D.G.

    1981-01-01

    Tritium suicide was shown to be a highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of ( 3 H) hypoxanthine for 5 or 10 min, followed by storage of 3 H-labelled cells at -70 0 C for 4-10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of ( 3 H)hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent Ksub(m) for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine. (orig.)

  7. Mutation of Chinese hamster cells by near-UV activation of promutagens

    International Nuclear Information System (INIS)

    Barnhart, B.J.; Cox, S.H.

    1980-01-01

    A tissue-culture assay for mutagenesis and cytotoxicity incorporating near ultraviolet (NUV) light activation of polyaromatic hydrocarbons (PAH) has been developed. Cultures of Chinese hamster cells (line CHO) growing in suspension culture were inoculated with benzo[a]pyrene (B[a]P), 7,12-dimethylbenzanthracene (DMBA) or shale-oil retort-water and exposed to light from a high-pressure mercury lamp fitted with a Corning NUV bandpass filter. This light source both permitted activation of PAH and the shale-oil water and precluded detectable damage to DNA. Neither the PAH nor the NUV alone had any effect on cell survival or mutation frequencies but the chemicals plus NUV were extremely effective in producing mutations to 6-thioguanine resistance (hgprt gene). (orig.)

  8. Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma.

    Science.gov (United States)

    Coppen, S R; Newsam, R; Bull, A T; Baines, A J

    1995-04-20

    The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.

  9. Effect of retinol and cigarette-smoke and condensate on dye-coupled intercellular communication between hamster tracheal epithelial cells

    NARCIS (Netherlands)

    Rutten, A.A.J.J.L.; Jongen, W.M.F.; Haan, L.H.J.de; Hendriksen, E.G.J.; Koeman, J.H.

    1988-01-01

    The dye-coupled intercellular communication across gap junctions in primary hamster tracheal epithelial cells has been studied in serum-free, hormone-supplemented medium. In the absence of vitamin A, non-cytotoxic concentrations of cigarette-smoke condensate (CSC) inhibited intercellular

  10. Embryo splitting

    Directory of Open Access Journals (Sweden)

    Karl Illmensee

    2010-04-01

    Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

  11. DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Olive, P.L.; Hilton, J.; Durand, R.E.

    1986-01-01

    Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this ''contact effect'' to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation

  12. A simple and reliable in vitro test system for the analysis of induced aneuploidy as well as other cytogenetic end-points using Chinese hamster cells

    International Nuclear Information System (INIS)

    Dulout, F.N.; Natarajan, A.T.

    1987-01-01

    Although aneuploidy is a serious human health problem, the experimental methodology devised until now to study the mechanisms involved in the induction of aneuploidy and for the screening of aneuploidy-inducing agents has not been so much employed to have the necessary validation. A procedure using primary cell cultures of Chinese hamster embryo cells grown on cover glasses is described. To avoid the excessive scattering and subsequent loss of chromosomes, a hypotonic treatment with a 0.17% sodium chloride solution, at room temperature, followed by in situ fixation has been standardized. This procedure improves the method through the reduction of the spontaneous frequency of aneuploid cells. Experiments carried out with cells treated with X-rays, X-rays plus caffeine, and the synthetic estrogen diethylstilbestrol (DES) demonstrated the accuracy of the system since the average chromosome number remained constant in spite of the induction of high frequencies of aneuploid cells. Moreover, the method allows for the analysis of other cytogenetic endpoints such as anaphase-telophase alterations, structural chromosome aberrations or sister chromatid exchanges. (author)

  13. Evidence for multiple repair pathways of double-strand DNA breaks in Chinese hamster cells

    International Nuclear Information System (INIS)

    Giaccia, A.J.; Weistein, R.; Stamato, T.D.; Roosa, R.

    1984-01-01

    XR-1 is a mutant of the Chinese hamster cell (CHO-K1) which is abnormally sensitive to killing by gamma rays in G/sub 1/ (D37 = 27 rads vs. 318 for parent) and early S phases of the cell cycle but has near normal resistance in late S and early G/sub 2/ (Somatic Cell Genetics, 9:165-173, 1983). Complementation studies between XR-1 and its parent indicate that this sensitivity to gamma rays is a recessive phenotype. Both the XR-1 and its parent cell are able to repair single strand DNA breaks. However, in comparison to its parental cell, the XR-1 cell is markedly deficient in the repair of double strand DNA breaks introduced by gamma irradiation during the sensitive G/sub 1/-early S period, while in the late S-G/sub 2/ resistant period the repair is similar in both cells. This correlation suggests that an unrepaired double strand DNA break is the lethal lesion and that at least two pathways for the repair of these lesions exist in mammalian cells

  14. Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis

    International Nuclear Information System (INIS)

    Klein, C.B.; Rossman, T.G.

    1990-01-01

    The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT - Chinese hamster V79 cells. Several gpt - cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. While spontaneous mutagenesis to gpt - occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and β-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus

  15. Effect of anolyte on growth and division of Chinese hamster cancerous cells

    Directory of Open Access Journals (Sweden)

    saeed Mohammadzadeh

    2009-04-01

    Full Text Available Background: At present, cancer can be controlled by chemotherapy, but unfortunately, this method has strong side effects and scientist try to reduce them using different substances. 2 kinds of activated water called anolyte and catholyte have electrochemical property and antibacterial and oxidative properties respectively. The aim of this research is to study the effect of anolyte on growth and division of cancerous cells. Materials and Methods: In this research, different concentration of anolyte, 1 . 7, 2, 5,8.3 and 10 percent of anolyte and control with 2 and 5 percent of serum physiologic were added on converted cell of Chinese hamster (line b11dii-FAF28 clone 237 in 12 plastic and 15 glass flasks. After adding, converted cell was counted with the help of hoemocytometer and microscope. Data of experiment analyzed and results compared by t test, as well as using Excell software their diagrams were drawn. Results: The results indicated that anolyte had significant effect on cancer cells. In concentration of 1.7% cell division was decreased but in concentration of 8.3 %, division of cancerous cells was blocked and cells were fixed. Conclusion: Considering the low amount of sodium chloride in anolyte, it seems that, this solution (Anolyte hasn’t side effects and advers effect on the cells body.

  16. Effect of dihydroxyanthraquinone (DHAQ) and radiation on the survival of cultured Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Kimler, B.F.

    1983-01-01

    Dihydroxyanthraquinone (DHAQ) is currently being tested as a cancer chemotherapeutic agent because of its structural similarity to Adriamycin (ADR) and other DNA-intercalating antibiotics. The interaction of DHAQ and ionizing radiation on the induction of cell lethality was investigated in Chinese hamster ovary cells in culture. In asynchronous populations of cells, DHAQ produced a slight enhancement of radiation-induced cell lethality as evidenced by changes in both shoulder and slope of the radiation dose-survival curves. However, DHAQ had no effect on either the extent or time course of recovery from sublethal radiation damage. In synchronous populations of cells treated at various times before or after selection in mitosis, the combination of DHAQ and radiation produced greater cell killing than that predicted based on simple additivity of effect, with a decided enhancement for cells treated during S phase. These results indicate that DHAQ is similar to other DNA-intercalating antibiotics in regard to the interaction with ionizing radiation to produce cell lethality

  17. A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

    KAUST Repository

    Hefzi, Hooman

    2016-11-23

    Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell-line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show that the metabolic resources in CHO are more than three times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses.

  18. Diethyldithiocarbamate enhancement of radiation and hyperthermic effects on Chinese hamster cells in vitro

    International Nuclear Information System (INIS)

    Lin, P.S.; Kwock, L.; Butterfield, C.E.

    1979-01-01

    To test the possibility of enhancing O 2 - toxicity during radiation therapy and/or hyperthermia, Chinese hamster cells (Don) were treated with the drug diethyldithiocarbamate (DDC) in vitro. This drug has been shown to inhibit, both in vitro and in vivo, the enzyme superoxide dismutase (SOD) which is responsible for eliminating the 0 2 - radical in cells. We observed that the cytocidal effect of DDC on Don cells is dependent on both DDC concentration and exposure time. After 8 to 10 days of incubation with 10 -9 M DDC, no change in cell survival was noted; however, incubation with 10 -4 M DDC produced a marked loss in colony formation. When DDC-treated cells were γ-irradiated, they did not survive as well as cells that were treated with either radiation or DDC alone. The combined effects of hyperthermia and DDC were dramatic. Cells treated 5 min at 43 0 C with 10 -4 M DDC or 10 min at 43 0 C with 10 -5 M DDC showed significant decrease in survival. These results suggest that DDC may be a potentially powerful sensitizing agent in tumor therapy, since there is increasing evidence that tumor cells have a lower level of superoxide dismutase

  19. Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures.

    Science.gov (United States)

    Nagle, W A; Soloff, B L; Moss, A J; Henle, K J

    1990-08-01

    Cultured Chinese hamster V79 fibroblast cells at the transition from logarithmic to stationary growth have been shown to undergo apoptosis (programmed cell death) after cold shock [B. L. Soloff, W. A. Nagle, A. J. Moss, Jr., K. J. Henle, and J. T. Crawford, Biochem. Biophys. Res. Commun. 145, 876-883 (1987)]. In this report, we show that about 95% of the cell population was susceptible to cold-induced apoptosis, and the amount of cell killing was dependent on the duration of hypothermia. Cells treated for 0-90 min at 0 degrees C exhibited an exponential survival curve with a D0 of 32 min; thus, even short exposures to the cold (e.g., 5 min) produced measurable cell killing. The cold-induced injury was not produced by freezing, because similar results were observed at 6 degrees C, and cell killing was not influenced by the cryoprotective agent dimethyl sulfoxide. Cold-induced apoptosis was inhibited by rewarming at 23 degrees C, compared to 37 degrees C, by inhibitors of macromolecular synthesis, such as cycloheximide, and by 0.8 mM zinc sulfate. The results suggest that apoptosis represents a new manifestation of cell injury after brief exposure to 0-6 degrees C hypothermia.

  20. Effects of laser uv-microirradiation (lambda=2573 A) on proliferation of Chinese hamster cells

    International Nuclear Information System (INIS)

    Cremer, C.; Cremer, T.; Zorn, C.; Schoeller, L.

    1976-01-01

    A laser uv-microbeam with a wavelength of 2573 A having a minimum spot diameter of approximately 0.5 μm was used to microirradiate interphase cells of a V-79 subline of Chinese hamster cells. The incident energy necessary to induce a significant decrease of proliferation was 30 to 60 times larger after microirradiation of cytoplasm as compared with microirradiation of nucleoplasm. The mean value of relative cell numbers 40 hr after irradiation as a function of incident energy did not differ whether the cells were microirradiated lying singly or together in small groups. Analysis of individual growth curves of singly lying cells microirradiated in the nucleoplasm with the same energy showed heterogeneous reactions. The incident energy per cell compatible with proliferation of about 50 percent of the cells after microirradiation of nucleoplasm was approximately 2 x 10 -3 ergs. From this value it is suggested that the energy density within the focus was in the region of several thousand ergs per square millimeter. Photochemical effects are thought to be the cause of growth disturbance, while thermal effects are excluded

  1. Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee-Chen, S.F.; Yu, C.T.; Jan, K.Y. (Academia Sinica, Taipei, (Taiwan). Institute of Zoology)

    1992-01-01

    Arsenite, an ubiquitous human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity and clastogenicity of UV light in mammalian cells. Arsenite may exert its co-genotoxic effects by inhibiting DNA repair. Results from alkaline sucrose gradient sedimentation show that arsenite did not accumulate UV-induced DNA strand breaks in Chinese hamster ovary (CHO) K1 cells as aphidicolin plus hydroxyurea (HU) did. These data indicate that arsenite did not inhibit the activity of DNA polymerase [alpha] in UV repair. Treatment with arsenite before UV irradiation slightly reduced the DNA strand breaks accumulated by cytosine [beta]-D-arabinofuranoside (AraC) plus HU. This effect implies that arsenite only slightly inhibited the incision of UV-induced DNA adducts. The low molecular weight DNA accumulated by post-UV incubation with AraC plus HU shifted to high molecular weight upon the incubation of cells in drug-free medium, but this shifting was prohibited by the presence of arsenite. This suggests that arsenite inhibited the rejoining of DNA strand breaks. When a pulse-chase labelling procedure was applied on UV-irradiated cells, the chain elongation of nascent DNA was strongly inhibited by post-incubation with arsenite. These data show that arsenite inhibited post-replication repair in UV-irradiated cells. Therefore, the steps inhibited by arsenite in UV-induced cells. Therefore, the steps inhibited by arsenite in UV-induced DNA repair in CHO K1 cells are different from human fibroblasts. (author).

  2. Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-hertz electromagnetic fields

    International Nuclear Information System (INIS)

    Stevenson, A.P.; Tobey, R.A.

    1985-01-01

    Potassium ion influx was measured by monitoring 42 KCl uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60-Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as loge of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4-7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K+ uptake could be attributed to the magnetic field. Additionally, intracellular K+ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K+ levels were observed between field-exposed and sham-exposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K+

  3. Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Lee-Chen, S.F.; Yu, C.T.; Jan, K.Y.

    1992-01-01

    Arsenite, an ubiquitous human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity and clastogenicity of UV light in mammalian cells. Arsenite may exert its co-genotoxic effects by inhibiting DNA repair. Results from alkaline sucrose gradient sedimentation show that arsenite did not accumulate UV-induced DNA strand breaks in Chinese hamster ovary (CHO) K1 cells as aphidicolin plus hydroxyurea (HU) did. These data indicate that arsenite did not inhibit the activity of DNA polymerase α in UV repair. Treatment with arsenite before UV irradiation slightly reduced the DNA strand breaks accumulated by cytosine β-D-arabinofuranoside (AraC) plus HU. This effect implies that arsenite only slightly inhibited the incision of UV-induced DNA adducts. The low molecular weight DNA accumulated by post-UV incubation with AraC plus HU shifted to high molecular weight upon the incubation of cells in drug-free medium, but this shifting was prohibited by the presence of arsenite. This suggests that arsenite inhibited the rejoining of DNA strand breaks. When a pulse-chase labelling procedure was applied on UV-irradiated cells, the chain elongation of nascent DNA was strongly inhibited by post-incubation with arsenite. These data show that arsenite inhibited post-replication repair in UV-irradiated cells. Therefore, the steps inhibited by arsenite in UV-induced cells. Therefore, the steps inhibited by arsenite in UV-induced DNA repair in CHO K1 cells are different from human fibroblasts. (author)

  4. Effects of 2'-chlorothymidine on Chinese hamster cells irradiated with x-rays and ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Murai, T; Kuwabara, M; Sato, F; Kubo, K; Itoh, T; Yoshii, G

    1985-06-01

    Effects of 2'-chlorothymidine (2'-Cl-TdR) and its mother compound, thymidine (TdR), on cell killing induced by X- and UV-irradiation have been investigated. Chinse hamster V-79 (TK/sup +/) cells as well as thymidine kinase deficient (TK/sup -/) variant cells, which were isolated from parental V-79 cells following stepwise treatment with BUdR, were incubated in a medium containing 2'-Cl-TdR and TdR after X- and UV-irradiation. In the TK/sup +/ cells, both 2'-Cl-TdR and TdR enhanced the killing efficiency of X-rays and ultraviolet light. On the other hand, in the TK/sup -/ cells, only 2'-Cl-TdR enhanced the killing efficiency of X- and UV-irradiation, and no effect of TdR was observed. These results suggest that phosphorylation of TdR by the enzyme is essential for its ability to modify radiation response, while the enhancement of cell killing by 2'-Cl-TdR must be explained by a mechanism at least partly independent of phosphorylation. (author).

  5. The effect of purine phosphonomethoxyalkyl derivatives on DNA synthesis in Cho Chinese hamster cells

    Energy Technology Data Exchange (ETDEWEB)

    Stetina, R [Institute of Experimental Medicine, Laboratory of Developmental Toxicology, Academy of Sciences of Czech Republic, 51783 Olesnice v Orlickych horach (Czech Republic); Votruba, I; Holy, A; Merta, A [Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic (Czech Republic)

    1994-12-31

    The inhibition of incorporation of {sup 3}H-thymidine and the changes of the rate of nascent DNA chain elongation were investigated in Cho Chinese hamster cells treated with (S)-(3-hydroxy-2-phosphonomethoxypropyl) (HPMP) and N-(2-phosphonomethoxyethyl) (PME) derivatives of adenine (A), guanine (G) and 2,6-diaminopurine (DAP). No direct correlation was observed in PME and HPMP derivatives between cytotoxicity, inhibition of {sup 3}H-thymidine incorporation and inhibition of nascent DNA chain elongation. The highest cytotoxicity and inhibition of DNA synthesis were caused by PMEG. The limited extent of inhibition of DNA elongation was encountered in the case of HPMPG and HPMPA. With PMEA, weak inhibition of elongation of DNA was observed only after a prolonged exposure (6 h). None of the investigated drugs induced DNA breaks. (author) 4 figs., 23 refs.

  6. UV-sensitivity of bromodeoxyuridine (BUdR)-substituted chromosomes in Chinese hamster cells

    International Nuclear Information System (INIS)

    Antoshchina, M.M.; Luchnik, N.V.

    1990-01-01

    Chinese hamster cells with chromosomes differently substituted for BUdR (TT-TT, TT-TB, TB-TB, TB-BB, where T is thymidine containing chromatid and B is BUdR substituted chromatid) were exposed to UV-light in phase G 2 and chromosome aberrations (mainly chromatid breaks) were analysed. Breaks frequency per chromosome was proportional to BUdR content. No breaks were found in TT-TT chromosomes. The frequency of breaks per TB chromatid was similar with TT-TB and TB-BB chromosomes. In TB-BB chromosomes, however, virtually no breaks occured in TB chromatids whereas in BB chromatids, their frequency was much higher than was expected

  7. Inhibition of DNA chain elongation in Chinese hamster cells by damage localized behind the replication fork

    Energy Technology Data Exchange (ETDEWEB)

    Ben-Hur, E [Israel Atomic Energy Commission, Beersheba. Nuclear Research Center-Negev; Hagan, M P [Armed Forces Radiobiology Research Inst., Bethesda, MD (USA)

    1984-05-01

    Chinese hamster fibroblasts were pulse labelled with 5-bromodeoxyuridine and exposed at time intervals (Tsub(i)) to near-ultraviolet (U.V.A.) light in the presence of a bisbenzimidazole derivative (Hoechst 33342). The sensitivity of the cells in terms of colony forming ability fluctuated depending on Tsub(i). Inhibition of DNA synthesis also depended on Tsub(i) and was maximal when Tsub(i)=O. Using the alkaline elution technique it was shown that the effect of a large dose of light was to inhibit both initiation and elongation of DNA chains. These effects were most pronounced for Tsub(i)=O. It is concluded that DNA damage in an active replicon can inhibit initiation of new replicons and that damage localized behind the replication fork can retard elongation of nascent DNA chains. This effect on chain elongation decreases with increased distance of the damage from the replication fork.

  8. Characterization of Chinese Hamster Ovary Cells Producing Coagulation Factor VIII Using Multi-omics Tools

    DEFF Research Database (Denmark)

    Kaas, Christian Schrøder

    The first public draft of a genome from Chinese hamster ovary (CHO) cells was published in 2011, an entire decade after the first draft of the human genome. This publication of a relevant CHO reference genome, in combination with the fact that the cost for DNA sequencing has dropped more than 10...... using omics tools. A wide range of methods were applied including whole-genome sequencing, targeted genome sequencing, mRNA sequencing, miRNA sequencing and mass spectrometry based shotgun proteomics on a number of clones in order to get a more holistic picture of the inner workings of these CHO...... transfectants. From the whole-genome sequencing of two CHO genomes (CHO DXB11 and the FVIII producing transfectant: F435) it was observed that roughly 20% of the genes in the genome were haploid and roughly 10% had a copy number of three or higher indicating extensive rearrangements compared to the Chinese...

  9. Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis

    International Nuclear Information System (INIS)

    Drillien, R.; Spehner, D.; Kirn, A.

    1978-01-01

    Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by uv irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective

  10. Effect of pepleomycin combined with irradiation on cultured Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Saito, Tsutomu

    1983-01-01

    The combined effect of pepleomycin (PEP), a bleomycin derivative, with irradiation was investigated on cultured Chinese hamster V79 cells. An additive effect was observed when PEP and irradiation were given simultaneously. A time interval between PEP (50μg/ml for one hour) and subsequent irradiation (10 Gy) increased the survival, and it became maximum when the time interval exceeded 2 hours. PEP-induced potentially lethal damage (PLD) was recovered when trysinization was delayed, and this recovery increased the survival. When PEP was given at a time interval after initial irradiation, the survival was decreased to below that following simultaneous treatment of the two modalities, and it became minimum when the time interval was 5 to 6 hours. Cells in ''G 2 -block'' induced by 10 Gy irradiation were partially synchronized, and cells in G 2 -M phase were more sensitive to PEP than those in S phase. It was considered that cells became more sensitive to PEP when they were irradiated 5 to 6 hours previously. However, cells recovery at any cell age when trypsinization was delayed. The benefit of a time interval between the two modalities was decreased by this recovery. (author)

  11. Inhibition of apoptosis using exosomes in Chinese hamster ovary cell culture.

    Science.gov (United States)

    Han, Seora; Rhee, Won Jong

    2018-05-01

    Animal cell culture technology for therapeutic protein production has shown significant improvement over the last few decades. Chinese hamster ovary (CHO) cells have been widely adapted for the production of biopharmaceutical drugs. In the biopharmaceutical industry, it is crucial to develop cell culture media and culturing conditions to achieve the highest productivity and quality. However, CHO cells are significantly affected by apoptosis in the bioreactors, resulting in a substantial decrease in product quantity and quality. Thus, to overcome the obstacle of apoptosis in CHO cell culture, it is critical to develop a novel method that does not have minimal concern of safety or cost. Herein, we showed for the first time that exosomes, which are nano-sized extracellular vesicles, derived from CHO cells inhibited apoptosis in CHO cell culture when supplemented to the culture medium. Flow cytometric and microscopic analyses revealed that substantial amounts of exosomes were delivered to CHO cells. Higher cell viability after staurosporine treatment was observed by exosome supplementation (67.3%) as compared to control (41.1%). Furthermore, exosomes prevented the mitochondrial membrane potential loss and caspase-3 activation, meaning that the exosomes enhanced cellular activities under pro-apoptotic condition. As the exosomes supplements are derived from CHO cells themselves, it is not only beneficial for the biopharmaceutical productivity of CHO cell culture to inhibit apoptosis, but also from a regulatory standpoint to diminish any safety concerns. Thus, we conclude that the method developed in this research may contribute to the biopharmaceutical industry where minimizing apoptosis in CHO cell culture is beneficial. © 2018 Wiley Periodicals, Inc.

  12. Effects of harman and norharman on spontaneous and ultraviolet light-induced mutagenesis in cultured Chinese hamster cells

    International Nuclear Information System (INIS)

    Chang, C.C.; Castellazzi, M.; Glover, T.W.; Trosko, J.E.

    1978-01-01

    Nontoxic concentrations of harman and norharman were tested in cultured Chinese hamster cells for their effects on DNA repair and mutagenesis. The following effects of harman were observed: (a) the survival of ultraviolet light- or x-ray-damaged cells was reduced; (b) the ultraviolet light-induced unscheduled DNA synthesis was slightly inhibited; and (c) the frequency of spontaneous or ultraviolet light-induced ouabain-resistant (ouar) or 6-thioguanine-resistant (6-TGr) mutations was reduced. Furthermore, the effect of harman on survival and mutagenesis was greater than that of norharman and was detected primarily in treatments in which cells were exposed to harman immediately following ultraviolet light irradiation. Our data clearly indicate that harman decreases the capacity to repair DNA damage and fix mutations in Chinese hamster cells, possibly because of the intercalation properties of this compound

  13. Expression of UV-irradiated adenovirus in normal and UV-sensitive Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Rainbow, A.J.

    1985-01-01

    The chinese hamster ovary (CHO) cell mutants UV-20, UV-24, and UV-41 are abnormally sensitive to UV and harbour various defects lin their ability to repair cellular DNA. This study has examined the expression of UV-irradiated AD2 in these cells. HCR of UV-irradiated Ad2, as measured by viral structural antigen (Vag) formation or progeny production, was found to be similar for the normal and the UV-sensitive CHO strains. UV-irradiation of Ad2 (1200 J/m/sup 2/) resulted in a delay of Vag expression of 18 hours in normal human fibroblasts, which is thought to reflect the time required for removal of UV-induced lesions from the DNA before viral DNA synthesis can proceed. However, a similar UV-irradiation of Ad2 did not result in a delay of Vag expression for infection of CHO cells, suggesting that UV-induced lesions in Ad2 DNA do not inhibit its replication in CHO cells. These results indicate a fundamental difference in the processing of UV-irradiated AD2-DNA in CHO as compared to human cells

  14. Genetic effects of the flavonols quercetin, kaempferol, and galangin on Chinese hamster ovary cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Carver, J.H. (Lawrence Livermore National Lab., Livermore, CA); Carrano, A.V.; MacGregor, J.T.

    1983-01-01

    The genotoxicity of selected flavonols was evaluated by multiple endpoints in Chinese hamster ovary (CHO) cells. Chromosomal aberrations, sister-chromatid exchange (SCE), and forward mutation at 4 gene loci were measured in a single population of cells exposed to quercetin, kaempferol, or galangin for 15 h with and without metabolic activation. The incidence of chromosomal aberrations was significantly increased by quercetin in the absence of activation and by kaempferol and galangin with and without activation. Flavanol treatment affected SCE and mutation at the hgprt, aprt, or Na/sup +//K/sup +/-ATPase loci only marginally, but significantly increased mutation frequencies at the tk locus. The response at the tk locus suggests that the CHO cells may behave similarly to L5178Y cells, in which the tk locus is thought to reflect chromosomal lesions in addition to point mutation. These results indicate that, at least under the conditions examined, flavonols induce chromosomal aberrations in CHO cells, but have little effect on point mutation or SCE.

  15. Spindle disturbances in human-hamster hybrid (AL) cells induced by mobile communication frequency range signals.

    Science.gov (United States)

    Schrader, Thorsten; Münter, Klaus; Kleine-Ostmann, Thomas; Schmid, Ernst

    2008-12-01

    The production of spindle disturbances in FC2 cells, a human-hamster hybrid (A(L)) cell line, by non-ionizing radiation was studied using an electromagnetic field with a field strength of 90 V/m at a frequency of 835 MHz. Due to the given experimental conditions slide flask cultures were exposed at room temperature in a microTEM (transversal electromagnetic field) cell, which allows optimal experimental conditions for small samples of biological material. Numerical calculations suggest that specific absorption rates of up to 60 mW/kg are reached for maximum field exposure. All exposure field parameters--either measured or calculable--are precisely defined and, for the first time, traceable to the standards of the SI system of physical units. Compared with co-incident negative controls, the results of two independently performed experiments suggest that exposure periods of time from 0.5 to 2 h with an electric field strength of 90 V/m are spindle acting agents as predominately indicated by the appearance of spindle disturbances at the ana- and telophase stages (especially lagging and non-disjunction of single chromosomes) of cell divisions. The spindle disturbances do not change the fraction of mitotic cells with increasing exposure time up to 2 h. Due to the applied experimental conditions an influence of temperature as a confounder parameter for spindle disturbances can be excluded.

  16. Diethyldithiocarbamate concentration effects and interactions with other cytotoxic agents on Chinese hamster cells (V79)

    International Nuclear Information System (INIS)

    Lin, P.S.; Quamo, S.; Ho, K.C.; Baur, K.

    1985-01-01

    A metal chelator, diethyldithiocarbamate (DDC) perturbs the chromosome condensation processes in dividing cells. The length of the metaphase chromosomes in Chinese hamster cells (V79) treated with 17.2 μg/ml of DDC for 2 hr is about half of that in untreated cells. However, concentrations of 1.7 μg or 172 μg/ml DDC apparently do not produce this effect. DDC at 17.2 μg/ml also disrupts spindle fibers. Bleomycin, but not mitomycin and cisplatin, added simultaneously with DDC can prevent the DDC effect on chromosomes. The cytotoxic effect of increasing concentrations of DDC can prevent the DDC effect on chromosomes. The cytotoxic effect of increasing concentrations of DDC to V79 cells incubated at 37 0 C exhibits a similar biphasic response. This concentration biphasic toxic effect is not altered when the cells are treated with DDC in combination with radiation, heat, or other cytotoxic drugs. These observations suggest that the different effects of DDC concentrations on chromosome condensation should be considered as one important modification factor for DDC related toxicity

  17. Mutagenicity of 8-methoxypsoralen and long-wave ultraviolet irradiation in V-79 Chinese hamster cells

    International Nuclear Information System (INIS)

    Burger, P.M.; Simons, J.W.I.M.

    1979-01-01

    The effect of 8-methoxypsoralen (8-MOP) and long-wave ultraviolet irradiation (UVA) on cell killing and mutation induction was studied in V-79 Chinese hamster cells. No effect was observed after treatment with 8-MOP alone (50 μg/ml, 4 h), UVA alone (9000 J/m 2 ), or 8-MOP metobolized by rat-liver microsomes. Combined treatment with 8-MOP and UVA induced both cell killing and mutation. This was also observed under conditins approaching patient treatment with PUVA photochemotherapy with respect to the concentration of 8-MOP in the skin and the amount of UVA received by the epidermal cells. A simple relation proved to apply for mutation induction under different treatment conditions: 5.5 X 10 -8 per J/m 2 per μg 8-MOP/ml. On this basis the mutation induction in dividing cells per session of PUVA-photochemotherapy amounts to 12.4 X 10 -5 , which is probably an over-estimation. (Auth.)

  18. Isolation and characterization of a Chinese hamster ovary cell mutant with altered regulation of phosphatidylserine biosynthesis

    International Nuclear Information System (INIS)

    Hasegawa, K.; Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1989-01-01

    We have screened approximately 10,000 colonies of Chinese hamster ovary (CHO) cells immobilized on polyester cloth for mutants defective in [14C]ethanolamine incorporation into trichloroacetic acid-precipitable phospholipids. In mutant 29, discovered in this way, the activities of enzymes involved in the CDP-ethanolamine pathway were normal; however, the intracellular pool of phosphorylethanolamine was elevated, being more than 10-fold that in the parental CHO-K1 cells. These results suggested that the reduced incorporation of [14C]ethanolamine into phosphatidylethanolamine in mutant 29 was due to dilution of phosphoryl-[14C]ethanolamine with the increased amount of cellular phosphorylethanolamine. Interestingly, the rate of incorporation of serine into phosphatidylserine and the content of phosphatidylserine in mutant 29 cells were increased 3-fold and 1.5-fold, respectively, compared with the parent cells. The overproduction of phosphorylethanolamine in mutant 29 cells was ascribed to the elevated level of phosphatidylserine biosynthesis, because ethanolamine is produced as a reaction product on the conversion of phosphatidylethanolamine to phosphatidylserine, which is catalyzed by phospholipid-serine base-exchange enzymes. Using both intact cells and the particulate fraction of a cell extract, phosphatidylserine biosynthesis in CHO-K1 cells was shown to be inhibited by phosphatidylserine itself, whereas that in mutant 29 cells was greatly resistant to the inhibition, compared with the parental cells. As a conclusion, it may be assumed that mutant 29 cells have a lesion in the regulation of phosphatidylserine biosynthesis by serine-exchange enzyme activity, which results in the overproduction of phosphatidylserine and phosphorylethanolamine as well

  19. Removal of radiation damage by subpopulations of plateau-phase Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Nelson, J.M.; Metting, N.F.; Braby, L.A.; Roesch, W.C.

    1987-01-01

    Specific cellular radiobiology studies are often required to test aspects of the mathematical models developed in the Radiation Dosimetry program. These studies are designed to determine whether specific mathematical expressions, which characterize the expected effect of biochemical mechanisms on observable biological responses, are consistent with the behavior of selected cell lines. Since these tests place stringent requirements on the cellular system, special techniques and culture conditions are required to minimize biological variability. The use of specialized cell populations is providing data on the extent of repair following low doses, and on the changes in the types of damage that can be repaired as the cell progresses toward mitosis. The stationary-phase Chinese hamster ovary (CHO) cells are composed primarily of G(1)-phase cells (83%), with the remainder comprising both G(2) and S phases. Removal of radiation damage by cells was studied in split-dose experiments. To date, we have observed no significant differences in cellular repair rate. This suggests, therefore, that each of the repair processes found in stationary-phase cells is cell-age independent. However, cellular radiation sensitivity does change rapidly and considerably as the cells progress from one phase to the next through the cell cycle. Since the rate of damage removal appears invariant, the change in survival must reflect the efficiency of producing that damage. The experimental data suggest that production of one or another sort of damage probably dominates during specific phases of the cell cycle, while the capacity for removal of all types of damage remains relatively constant

  20. Effects of turmeric and its active principle, curcumin, on bleomycin-induced chromosome aberrations in Chinese hamster ovary cells

    OpenAIRE

    Araújo, Maria Cristina P.; Dias, Francisca da Luz; Kronka, Sergio N. [UNESP; Takahashi, Catarina S.

    1999-01-01

    Naturally occurring antioxidants have been extensively studied for their capacity to protect organisms and cells from oxidative damage. Many plant constituents including turmeric and curcumin appear to be potent antimutagens and antioxidants. The effects of turmeric and curcumin on chromosomal aberration frequencies induced by the radiomimetic agent bleomycin (BLM) were investigated in Chinese hamster ovary (CHO) cells. Three concentrations of each drug, turmeric (100, 250 and 500 mg/ml) and ...

  1. Stem cell research on other worlds, or why embryos do not have a right to life.

    Science.gov (United States)

    Blackford, R

    2006-03-01

    Anxieties about the creation and destruction of human embryos for the purpose of scientific research on embryonic stem cells have given a new urgency to the question of whether embryos have moral rights. This article uses a thought experiment involving two possible worlds, somewhat removed from our own in the space of possibilities, to shed light on whether early embryos have such rights as a right not to be destroyed or discarded (a "right to life"). It is argued that early embryos do not have meaningful interests or any moral rights. Accordingly, claims about the moral rights of embryos do not justify restrictions on stem cell research.

  2. Embryos, Clones, and Stem Cells: A Scientific Primer

    Directory of Open Access Journals (Sweden)

    Kenyon S. Tweedell

    2004-01-01

    Full Text Available This article is intended to give the nonspecialist an insight into the nuances of “clones”, cloning, and stem cells. It distinguishes embryonic and adult stem cells, their normal function in the organism, their origin, and how they are recovered to produce stem cell lines in culture. As background, the fundamental processes of embryo development are reviewed and defined, since the manipulation of stem cell lines into desired specialized cells employs many of the same events. Stem cells are defined and characterized and shown how they function in the intact organism during early development and later during cell regeneration in the adult. The complexity of stem cell recovery and their manipulation into specific cells and tissue is illustrated by reviewing current experimentation on both embryonic and adult stem cells in animals and limited research on human stem cell lines. The current and projected use of stem cells for human diseases and repair, along with the expanding methodology for the recovery of human embryonic stem cells, is described. An assessment on the use of human embryonic stem cells is considered from ethical, legal, religious, and political viewpoints.

  3. Modulation of in vitro transformation and the early and late modes of DNA replication of uv-irradiation Syrian hamster cells by caffeine

    International Nuclear Information System (INIS)

    Doniger, J.; DiPaolo, J.A.

    1981-01-01

    The effect of caffeine on post-uv DNA replication was studied to determine its relevance to carcinogenesis. The level of uv-induced transformed colonies of Syrian hamster embryo cells (HEC) was increased up to fivefold when caffeine was added to cells between 0 and 6 h post-uv. The greatest increase was observed when the interval between uv irradiation and caffeine addition was 4 h. Two modes of DNA replication occurred after uv irradiation. During the early mode (0 to 3 h post-uv) the size of nascent strands, as measured by alkaline sucrose sedimentation, was smaller than those in nonirradiated cells, whereas during the late mode they recovered to normal size. Caffeine inhibited the rate of elongation of nascent strands during the early mode. When caffeine was added immediately after uv irradiation, the conversion of the early mode to the late mode was inhibited. Studies on the effects of caffeine have now been extended to the late mode. While caffeine has little effect with the fd elements beginning from the 10th day after irradiation is connected with their proliferation but not with the migration out from lymphoid organs

  4. Hyperthermic radiosensitization of synchronous Chinese hamster cells: relationship between lethality and chromosomal aberrations

    International Nuclear Information System (INIS)

    Dewey, W.C.; Sapareto, S.A.; Betten, D.A.

    1978-01-01

    Synchronous Chinese hamster cells in vitro were obtained by mitotic selection. The cells were heated at 45.5 0 C for 4 min in mitosis, 11 min in G 1 , or 7 min in S sphase and then x-irradiated immediately thereafter. Colony survival from heat alone was 0.30 to 0.45, and the frequency of chromosomal aberrations induced by heat was 0.00, 0.14, or 0.97 for heat treatments during M, G 1 , or S, respectively. As shown previously, lethality from hyperthermia alone is due to chromosomal aberrations only when the cells are heated during S phase. The log survival (D 0 /sup approximately/ = 80 rad) and aberration frequency curves for cells irradiated during mitosis were linear, and the only effect of hyperthermia was to shift the curves in accord with the effect from heat alone. Thus, hyperthermia did not radiosensitize the mitotic cells. The cells irradiated in G 1 were more resistant (D 0 /sup approximately/ = 100 rad) than those irradiated in mitosis, and the survival and aberration frequency curves both had shoulders. The primary effect of hyperthermia was to greatly reduce the shoulders of the curves and to increase the slopes by about 23%. The cells irradiated in S were the most resistant (D 0 /sup approximately/ = 140 rad), and the survival and aberration frequency curves both had large shoulders. For both end points of lethality and chromosomal aberrations, heat selectively radiosensitized S-phase cells relative to G 1 cells by removing most of the shoulder and increasing the slope by about 45%. For cells treated in G 1 or S, the increase in radiosensitization following hyperthermia can be accounted for by an increase in the frequency of chromosomal aberrations

  5. Specific estrogen-induced cell proliferation of cultured Syrian hamster renal proximal tubular cells in serum-free chemically defined media

    International Nuclear Information System (INIS)

    Oberley, T.D.; Lauchner, L.J.; Pugh, T.D.; Gonzalez, A.; Goldfarb, S.; Li, S.A.; Li, J.J.

    1989-01-01

    It has long been recognized that the renal proximal tubular epithelium of the hamster is a bona fide estrogen target tissue. The effect of estrogens on the growth of proximal tubule cell explants and dissociated single cells derived from these explant outgrowths has been studied in culture. Renal tubular cells were grown on a PF-HR-9 basement membrane under serum-free chemically defined culture conditions. At 7-14 days in culture, cell number was enhanced 3-fold in the presence of either 17β-estradiol or diethylstilbestrol. A similar 3-fold increase in cell number was also seen at 1 nM 17β-estradiol in subcultured dissociated single tubular cells derived from hamster renal tubular explant outgrowths at 21 days in culture. Concomitant exposure of tamoxifen at 3-fold molar excess in culture completely abolished the increase in cell number seen with 17β-estradiol. The proliferation effect of estrogens on proximal tubular cell growth appears to be species specific since 17β-estradiol did not alter the growth of either rat or guinea pig proximal tubules in culture. In addition, at 7-10 days in culture in the presence of 17β-estradiol, [ 3 H]thymidine labeling of hamster tubular cells was enhanced 3-fold. These results clearly indicate that estrogens can directly induce primary epithelial cell proliferation at physiologic concentrations and provide strong additional evidence for an important hormonal role in the neoplastic transformation of the hamster kidney

  6. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents

    International Nuclear Information System (INIS)

    Kaina, B.; Lohrer, H.; Karin, M.; Herrlich, P.

    1990-01-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions

  7. Isolation and characterization of a radiosensitive Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Fuller, L.F.

    1987-01-01

    A x-ray sensitive Chinese hamster ovary cell line was isolated using a semi-automated procedure in which mutagenized CHO cells were allowed to form colonies on top of agar, x-irradiated, then photographed at two later times. Comparison of the photographs allowed the identification of colonies which displayed significant growth arrest. One of the colonies identified in this manner produced a stable, radiosensitive line. This cell line is normal in x-ray induced inhibition of DNA synthesis, and single- and double-strand break repair, and is moderately sensitive to ethyl methane sulfonate and UV light. The sensitive line performs only half as much x-ray-induced repair replication as the parental line and this deficiency is believed to be the primary cause of its radiosensitivity. The sensitive line produces significantly higher numbers of x-ray-induced chromosome and chromatid aberrations including chromatid aberrations following exposure during the G 1 phase of the cell cycle. The line is hypomutable compared to the parental line with x-ray exposure inducing only one-third as many 6-thioguanine resistant colonies

  8. Interactive cytotoxicity of etoposide and radiation on cultured Chinese hamster V-79 cells

    International Nuclear Information System (INIS)

    Saito, Tsutomu; Shimada, Yuji; Kamata, Rikisaburo

    1989-01-01

    Etoposide is a semisynthetic derivative of podophyllotoxin and is an active antitumor agent. The interactive cytotoxic effect of Etoposide and radiation was investigated using cultured Chinese hamster V-79 cells. The surviving fraction of the cells was reduced by only 20%, when the cells were exposed to 5μg/ml of Etoposide for 30 min. Etoposide at this concentration reduced the width of the shoulder of the radiation survival curve. The change became more significant with increase in the concentration of Etoposide. The Dqs (quasithreshold doses) of the radiation survival curves were 5.39, 3.28, 2.13 and 0.54Gy, although the Dos (37% dose slopes) of the radiation survival curves were 2.55, 2.49, 2.39 and 2.18 Gy, when combination treatment with radiaiton and 0, 5, 10 and 20 μg/ml of Etoposide, respectively, was carried out. The cytotoxic effect became increased when fractional treatments with Etoposide and radiation were performed. The results obtained suggest that the mechanism of the interactive cytotoxic effect of this combination treatment involves a reciprocal action of Etoposide and sublethal damage by the radiation to the cells. (author)

  9. Chromosome studies on bone marrow cells of chinese hamsters fed a radiosterilized diet

    International Nuclear Information System (INIS)

    Renner, H.W.

    1977-01-01

    Metaphase preparations of chromosomes from bone marrow cells of Chinese hamsters were examined for mutagenic effects following the feeding of a radiosterilized diet. No increase in the incidence of structural chromosomal aberrations was observed. As far as numerical aberrations were concerned, the proportion of cells with polyploidy increased to between 4 to 5 times the control level, irrespective of the moisture content of the diet. This polyploidy effect occurred very early, being detectable within 24 h, if the diet fed had been irradiated with an absorbed dose of 4.5x10 6 rad. The incidence of polyploidy remained below 0.5%, however, nor did it rise with higher radiation doses. When the feeding of the irradiated diet was stopped, the proportion of polyploid cells returned to the control level within a maximum of 6 weeks. If the diet was stored (initially) for 6 weeks following irradiation before being fed to the animals no increase in the number of polyploid cells was noted. These results are not interpreted as a mutagenic effect of the irradiated diet. (author)

  10. Ferritin-iron increases killing of Chinese hamster ovary cells by X-irradiation

    International Nuclear Information System (INIS)

    Nelson, J.M.; Stevens, R.G.

    1992-01-01

    Stationary-phase Chinese hamster ovary cells were cultured in medium containing ferritin (∼19% iron by weight) added at concentrations ranging from 0 to 128 μg/ml. One set of cultures was unirradiated, another set exposed to 4.0 Gy of X-ray. Clonogenic cell survival was assessed in each set of cultures. In the absence of added ferritin, 4.0 Gy killed approximately 50% of the cells. In the absence of radiation, ferritin was not toxic at less than 48 μg/ml; above 48 μg/ml, toxicity increased with concentration. Apoferritin was not toxic at any concentration tested (up to 1000 μg/ml). Although 32 μg/ml ferritin, reflecting only a 3-6 fold increase in iron concentration over normal serum, was not toxic, it reduced survival of X-irradiated cells by an additional 75%. These results indicate that a sublethal concentration of ferritin can be a potent radiosensitizer. (Author)

  11. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents

    Energy Technology Data Exchange (ETDEWEB)

    Kaina, B.; Lohrer, H.; Karin, M.; Herrlich, P. (Kernforschungszentrum Karlsruhe, Karlsruhe (Germany, F.R.))

    1990-04-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions.

  12. Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.

    Science.gov (United States)

    Kaina, B; Lohrer, H; Karin, M; Herrlich, P

    1990-01-01

    Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions. Images PMID:2320583

  13. Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuscoe, J.C.; O' Neill, J.P.; Machanoff, R.; Hsie, A.W.

    1982-01-01

    An assay is described for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10/sup 6/ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10..mu..M) is then added directly to the growing culture and AS-resistant (AS/sup r/) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT/sup -/ clones. The AS/sup r/ phenotype is characterized both physiologically and biochemically. All AS/sup r/ clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT.

  14. Gamma radiation inhibits the appearance of induced ornithine decarboxylase activity in Chinese hamster cells

    International Nuclear Information System (INIS)

    Ben-Hur, E.; Heimer, Y.M.; Riklis, E.

    1981-01-01

    Ornithine decarboxylase activity of Chinese hamster cells (ODC, EC 4.1.1.17) can be induced in plateau phase by change of medium. Exposure of the cells to gamma radiation before induction reduces the amount of ODC activity induced. The dose-response curve is exponential with a D 0 of 106 krad. Exposure of BUdR-substituted cells is more effective in reducing ODC induction at high doses, with a D 0 of 38 krad. Cells can recover from the reduction incurred by 74 krad if enzyme induction is delayed for 2 hours after exposure. Treatment of the cells with psoralen-plus-light completely inhibits RNA synthesis without affecting protein synthesis (Heimer, Ben-Hur and Riklis 1977, 1978). Using this procedure it is shown that the effect of gamma radiation on inducible ODC activity is due not only to DNA damage but also involves a post-transcriptional effect. This conclusion is supported by employing a heat shock to inhibit protein synthesis prior to gamma-irradiation of log-phase cells. In such cells the increased activity of ODC upon transfer to 37 0 C is due primarily to enzyme synthesis using pre-existing RNA species during the first few hours. A low concentration of actinomycin D, which inhibits rRNA synthesis, applied during the recovery period, prevents the recovery of the cells' capacity for maximal ODC induction. This may indicate that, in order to recover, the cells have to repair damage to the ribosomes as well as to DNA. (author)

  15. Cell lineages of the embryo of the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Deppe, U; Schierenberg, E; Cole, T; Krieg, C; Schmitt, D; Yoder, B; von Ehrenstein, G

    1978-01-01

    Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock--i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution video recording.

  16. Inhibition of DNA replication by ozone in Chinese Hamster V79 cells

    International Nuclear Information System (INIS)

    Rasmussen, R.E.

    1986-01-01

    DNA replication in Chinese hamster lung fibroblasts, line V79, was depressed in a dose-dependent manner over an ozone concentration range of 1-10 ppm. When the cells were exposed for 1 h at concentrations up to 6 ppm, the rate of DNA replication, as measured by [ 3 H]thymidine incorporation, declined further during a 3-h period immediately following exposure. At higher ozone concentrations, at which more than 99.9% of the cells were killed, no further decline in DNA replication was seen beyond that immediately following exposure. Cultures exposed for 1 h to 10 mM ethyl methanesulfonate or to 10 J/m 2 of ultraviolet (UV) light showed a similar progressive decline in the rate of DNA replication. The inhibition of DNA replication by ozone resembled that seen after exposure of cells to chemical mutagens or radiation and did not resemble the inhibition produced by metabolic poisons. The results may indicate that ozone or its reaction products interact directly with DNA in a way that inhibits replication

  17. Cell inactivation and chromosomal aberrations induced by X-rays and fast neutrons in cells of the Chinese hamster. 1

    International Nuclear Information System (INIS)

    Tolkendorf, E.

    1979-01-01

    Asynchronously grown cultures of Chinese hamster cells V79-4 were irradiated in suspension with 180 kV X-rays and fast neutrons (average energy of 6.2 MeV). The damage was assessed by measuring cell survival and frequencies of chromosome aberrations in the first post-irradiation metaphases. The experimental data for survival and chromosome aberrations were fitted by computer programmes. From the fitted curves the relative biological effectiveness (RBE) of fast neutrons was calculated. The RBE shows a similar dose dependence for killed and aberrant cells. The RBE decreases with increasing dose and amounts to approximately 5 for both effects for small neutron doses. The highest RBE is found for asymmetrical chromosomal exchanges and is dependent on the neutron dose, too. However, for isochromatid deletions the RBE is dose independent with a value of 3.6. (author)

  18. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

    International Nuclear Information System (INIS)

    Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1986-01-01

    Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with 32 Pi and L-[U- 14 C]serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% of that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells

  19. Effect of various 3H-thymidine concentrations on the kinetics of chinese hamster cell division

    International Nuclear Information System (INIS)

    Yuzhakov, V.V.; Lychev, V.A.

    1985-01-01

    A study of the asynchronous culture of Chinese hamster fibroblasts by autoradiography has shown that the pulse (15 min) incorporation of 3 H-thymidine in nuclear DNA influences the kinetics of labelled cell proliferation. The results obtained suggest that one of the early biological effects of the pulse incorporation of 3 H-thymidine is a delay in the occurrence of the first mitosis. With the concentration of 3 H-thymidine 37 kBq/ml the slowing down of the movement of labelled cells in the cycle is detected by a shift and overlapping of waves of labelled and unlabelled mitotic cells. In an increase of the concentration up to 370-925 kBq/ml the pattern of the curves of labelled mitotic cells is distorted. These distortions are well interpreted by the nature of change of the index of labelled and unlabelled mitotic cells. After an increase in 3 H-thymidine concentration from 37 up to 370-925 kBq/ml the mitotic activity of cells labelled at the end of S-phase decreases from 1 to o0.6-0.1% respectively. With the concentration of 925 kBq/ml for these cells incorporating 3 H-thymidine at the end of S-phase, a delay of the entry into mitosis reaches 6-8 h. Autoradiography data with assessment of granule density suggest that mitotic activity and the period of delay in the occurrence of mitosis depend on the dose of irradiation with intranuclear tritium

  20. Diversity in host clone performance within a Chinese hamster ovary cell line.

    Science.gov (United States)

    O'Callaghan, Peter M; Berthelot, Maud E; Young, Robert J; Graham, James W A; Racher, Andrew J; Aldana, Dulce

    2015-01-01

    Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc-fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool-produced Mab and etanercept (by N-glycan ultra performance liquid chromatography (UPLC) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N-glycan micro-heterogeneity and etanercept N and O-linked macro-heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers.

  1. Endocardial tip cells in the human embryo - facts and hypotheses.

    Directory of Open Access Journals (Sweden)

    Mugurel C Rusu

    Full Text Available Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43-56 days were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin, CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.

  2. Asymmetries in Cell Division, Cell Size, and Furrowing in the Xenopus laevis Embryo.

    Science.gov (United States)

    Tassan, Jean-Pierre; Wühr, Martin; Hatte, Guillaume; Kubiak, Jacek

    2017-01-01

    Asymmetric cell divisions produce two daughter cells with distinct fate. During embryogenesis, this mechanism is fundamental to build tissues and organs because it generates cell diversity. In adults, it remains crucial to maintain stem cells. The enthusiasm for asymmetric cell division is not only motivated by the beauty of the mechanism and the fundamental questions it raises, but has also very pragmatic reasons. Indeed, misregulation of asymmetric cell divisions is believed to have dramatic consequences potentially leading to pathogenesis such as cancers. In diverse model organisms, asymmetric cell divisions result in two daughter cells, which differ not only by their fate but also in size. This is the case for the early Xenopus laevis embryo, in which the two first embryonic divisions are perpendicular to each other and generate two pairs of blastomeres, which usually differ in size: one pair of blastomeres is smaller than the other. Small blastomeres will produce embryonic dorsal structures, whereas the larger pair will evolve into ventral structures. Here, we present a speculative model on the origin of the asymmetry of this cell division in the Xenopus embryo. We also discuss the apparently coincident asymmetric distribution of cell fate determinants and cell-size asymmetry of the 4-cell stage embryo. Finally, we discuss the asymmetric furrowing during epithelial cell cytokinesis occurring later during Xenopus laevis embryo development.

  3. Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.

    Science.gov (United States)

    Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A; Esko, Jeffrey D; Linhardt, Robert J; Sharfstein, Susan T

    2012-03-01

    Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

    DEFF Research Database (Denmark)

    Svarcova, Olga; Dinnyes, A.; Polgar, Z.

    2009-01-01

    displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

  5. Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells

    International Nuclear Information System (INIS)

    Karentz, D.; Cleaver, J.E.

    1986-01-01

    Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia

  6. Laser microirradiation of Chinese hamster cells at wavelength 365 nm: effects of psoralen and caffeine

    International Nuclear Information System (INIS)

    Cremer, T.; Peterson, S.P.; Cremer, C.; Berns, M.W.

    1981-01-01

    Cells of a V79 subline of the Chinese hamster were microirradiated at wavelength 365 nm in the presence of the psoralen derivative, trioxsalen. Microirradiation was accomplished by a pulsed argon laser microbeam either in anaphase or in interphase 3 h after mitosis. Inhibition of clonal growth and formation of micronuclei at the first postirradiation mitosis were observed after microirradiation of anaphase chromosomes and of small parts of the interphase nucleus. Microirradiation of the cytoplasm beside the interphase nucleus or between the sets of chromosomes moving apart from each other in anaphase did not produce these effects. Anaphase experiments showed that only the daughter cell which received microirradiated chromatin exhibited an abnormal growth pattern. Most interestingly, shattering of the whole chromosome complement could be induced by microirradiation of small parts of the interphase nucleus and post-treatment with caffeine. Since microirradiation of chromatin in the absence of psoralen was not effective, we consider formation of psoralen photoadducts to nucleic acids in microirradiated chromatin to be the specific cause of the effects. We suggest that DNA photolesions in chromosome segments present in the microirradiated part of the nucleus can induce shattering of all the chromosomes in the microirradiated nucleus. Several possibilities are discussed to explain this unexpected finding

  7. Ascorbate enhances u.v.-mutagenesis in E. coli but inhibits it in Chinese hamster cells

    International Nuclear Information System (INIS)

    Rossman, T.G.; Klein, C.B.; Naslund, M.

    1986-01-01

    Ascorbic acid (vitamin C) causes an increase in the mutation frequency of u.v.-irradiated Escherichia coli WP2. The enhancement occurs at all u.v. fluences, and is dependent upon the ascorbate concentration in the medium. A maximum effect (approx. 8- to 13-fold) is seen at 100-150 μg/ml, although some enhancement can be seen even at 10 μg/ml. The comutagenic effect of ascorbate with u.v. in E. coli is dependent upon peptone, a constituent of nutrient broth. The enhancement of u.v.-mutagenesis by ascorbate is absent in strains WP2sub(s) (uvrA) amd WP6 (polA), suggesting that ascorbate affects the repair of pyrimidine dimers. The opposite results are observed for u.v.-mutagenesis in Chinese hamster V79 cells. The presence of ascorbate (50 μg/ml) during u.v. irradiation does not enhance the u.v. effect, but rather decreases it approx. 30%. These results are discussed with regard to differences in the mechanism of u.v.-mutagenesis and DNA repair in bacterial and mammalian cells. (author)

  8. The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

    Directory of Open Access Journals (Sweden)

    Xiangyi Kong

    Full Text Available Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI treatment cycles, n = 799 were classified as follows: less than 5 cells (10C; n = 42. Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In 10C embryos increased compared to 7-8C embryos (45.8%, 33.3% vs. 11.1%, respectively. In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas 10C. In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

  9. Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

    DEFF Research Database (Denmark)

    Smith, Steven D.; Soloy, Eva; Kanka, Jiri

    1996-01-01

    Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo....... NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...

  10. Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Davis, J.M.; Arakawa, T.; Strickland, T.W.; Yphantis, D.A.

    1987-01-01

    Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I - and acrylamide but not by Cs + . On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability

  11. Mutagenicity and antimutagenicity of Baccharis dracunculifolia extract in chromosomal aberration assays in Chinese hamster ovary cells.

    Science.gov (United States)

    Munari, Carla Carolina; Resende, Flávia Aparecida; Alves, Jacqueline Morais; de Sousa, João Paulo; Bastos, Jairo Kenupp; Tavares, Denise Crispim

    2008-09-01

    Baccharis dracunculifolia De Candole (Asteraceae), a native plant from the Brazilian "cerrado", is widely used in folk medicine as an anti-inflammatory agent and for the treatment of gastrointestinal diseases. B. dracunculifolia has been described as the most important plant source of propolis in southeastern Brazil, which is called green propolis due to its color. The aim of the present study was to evaluate the mutagenic and antimutagenic effects of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE) on Chinese hamster ovary cells. On one hand, the results showed a significant increase in the frequencies of chromosome aberrations at the highest Bd-EAE concentration tested (100 microg/mL). On the other hand, the lowest Bd-EAE concentration tested (12.5 micro/mL) significantly reduced the chromosome damage induced by the chemotherapeutic agent doxorubicin. The present results indicate that Bd-EAE has the characteristics of a so-called Janus compound, that is, Bd-EAE is mutagenic at higher concentrations, whereas it displays a chemopreventive effect on doxorubicin-induced mutagenicity at lower concentrations. The constituents of B. dracunculifolia responsible for its mutagenic and antimutagenic effects are probably flavonoids and phenylpropanoids, since these compounds can act either as pro-oxidants or as free radical scavengers depending on their concentration.

  12. Induction of stable protein-deoxyribonucleic acid adducts in Chinese hamster cell chromatin by ultraviolet light

    International Nuclear Information System (INIS)

    Strniste, G.F.; Rall, S.C.

    1976-01-01

    Ultraviolet (uv)-light-mediated formation of protein-DNA adducts in Chinese hamster cell chromatin was investigated in an attempt to compare chromatin alterations induced in vitro with those observed in vivo. Three independent methods of analysis indicated stable protein-DNA associations: a membrane filter assay which retained DNA on the filter in the presence of high salt-detergent; a Sepharose 4B column assay in which protein eluted coincident with DNA; and a CsCl density gradient equilibrium assay which showed both protein and DNA banding at densities other than their respective native densities. Treatment of the irradiated chromatin with DNase provided further evidence that protein--DNA and not protein-protein adducts were being observed in the column assay. There is a fluence-dependent response of protein-DNA adduct formation when the chromatin is irradiated at low ionic strength and is linear for protein over the range studied. When the chromatin is exposed to differing conditions of pH, ionic strength, or divalent metal ion concentration, the quantity of adduct formed upon uv irradiation varies. Susceptibility to adduct formation can be partially explained in terms of the condensation state of the chromatin and other factors such as rearrangement, denaturation, and dissociation of the chromatin components. Besides providing information on the biological significance of these types of uv-induced lesions, this technique may be useful as a probe of chromatin structure

  13. The effect of dexamethasone on the radiation survival response and misonidazole-induced hypoxic-cell cytotoxicity in Chinese hamster cells V-79-753B in vitro

    International Nuclear Information System (INIS)

    Millar, B.C.; Jinks, S.

    1981-01-01

    Overnight exposure of Chinese hamster cells, V-79-753B, to microgram quantities of the synthetic corticosteroid, dexamethasone, resulted in a decrease in sensitivity towards radiation, both in air and in hypoxia. The effect was dose-modifying and the oxygen enhancement ratio did not change appreciably. Similarly, when dexamethasone-treated hypoxic cells were irradiated in the presence of misonidazole, a hypoxic cell radiosensitizer, there was a decrease in radiation sensitivity compared with untreated hypoxic cells irradiated with misonidazole. (author)

  14. A mechanistic study on the effect of dexamethasone in moderating cell death in Chinese Hamster Ovary cell cultures.

    Science.gov (United States)

    Jing, Ying; Qian, Yueming; Ghandi, Mahmoud; He, Aiqing; Borys, Michael C; Pan, Shih-Hsie; Li, Zheng Jian

    2012-01-01

    Dexamethasone (DEX) was previously shown (Jing et al., Biotechnol Bioeng. 2010;107:488-496) to play a dual role in increasing sialylation of recombinant glycoproteins produced by Chinese Hamster Ovary (CHO) cells. DEX addition increased sialic acid levels of a recombinant fusion protein through increased expression of α2,3-sialyltransferase and β1,4-galactosyltransferase, but also decreased the sialidase-mediated, extracellular degradation of sialic acid through slowing cell death at the end of the culture period. This study examines the underlying mechanism for this cytoprotective action by studying the transcriptional response of the CHO cell genome upon DEX treatment using DNA microarrays and gene ontology term analysis. Many of those genes showing a significant transcriptional response were associated with the regulation of programmed cell death. The gene with the highest change in expression level, as validated by Quantitative PCR assays with TaqMan® probes and confirmed by Western Blot analysis, was the antiapoptotic gene Tsc22d3, also referred to as GILZ (glucocorticoid-induced leucine zipper). The pathway by which DEX suppressed cell death towards the end of the culture period was also confirmed by showing involvement of glucocorticoid receptors and GILZ through studies using the glucocorticoid antagonist mifepristone (RU-486). These findings advance the understanding of the mechanism by which DEX suppresses cell death in CHO cells and provide a rationale for the application of glucocorticoids in CHO cell culture processes. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  15. Permeabilization of ultraviolet-irradiated chinese hamster cells with polyethylene glycol and introduction of ultraviolet endonuclease from Micrococcus luteus

    International Nuclear Information System (INIS)

    Yarosh, D.B.; Setlow, R.B.

    1981-01-01

    Chinese hamster V-79 cells were made permeable by treatment with polyethylene glycol and then incubated with a Micrococcus luteus extract containing ultraviolet-specific endonuclease activity. This treatment introduced nicks in irradiated, but not in unirradiated, deoxyribonucleic acid. The nicks remained open for at least 3 h; there was no loss of endonuclease-sensitive sites, and no excision of dimers as measured by chromatography was detected. In addition, there was no increase in ultraviolet resistance in treated cells. This suggests that the absence of a significant amount of excision repair in rodent cells is due to the lack of both incision and excision capacity

  16. The cytogenetic effect of radiation and postirradiation treatment of Chinese hamster cells with arabinoside cytosine and hydroxyurea

    International Nuclear Information System (INIS)

    Elisova, T.V.; Stavrakova, N.M.; Feoktistova, T.P.

    1988-01-01

    A two-hour treatment of Chinese hamster cells at the G 1 stage of the cell cycle with arabinoside cytocine combined with hydroxyurea after X-irradiation (50-300 cGy) produced a 2- to 4-fold increase in the frequency of chromosome aberrations. The mitotic selection method was used to synchronize the cells. The potentiating effect of inhibitors was estimated by the yield of centric exchanges decreased with increasing radiation dose. It is suggested that DNA repair processes determining a linear component of the dose-response curve are modified within the dose-range under study

  17. Aligned, isotropic and patterned carbon nanotube substrates that control the growth and alignment of Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Abdullah, Che Azurahanim Che; Asanithi, Piyapong; Brunner, Eric W; Jurewicz, Izabela; Bo, Chiara; Sear, Richard P; Dalton, Alan B [Department of Physics and Surrey Materials Institute, University of Surrey, Guildford, Surrey GU2 7XH (United Kingdom); Azad, Chihye Lewis; Ovalle-Robles, Raquel; Fang Shaoli; Lima, Marcio D; Lepro, Xavier; Collins, Steve; Baughman, Ray H, E-mail: r.sear@surrey.ac.uk [Alan G MacDiarmid NanoTech Institute, The University of Texas at Dallas, Richardson, TX 75080-3021 (United States)

    2011-05-20

    Here we culture Chinese hamster ovary cells on isotropic, aligned and patterned substrates based on multiwall carbon nanotubes. The nanotubes provide the substrate with nanoscale topography. The cells adhere to and grow on all substrates, and on the aligned substrate, the cells align strongly with the axis of the bundles of the multiwall nanotubes. This control over cell alignment is required for tissue engineering; almost all tissues consist of oriented cells. The aligned substrates are made using straightforward physical chemistry techniques from forests of multiwall nanotubes; no lithography is required to make inexpensive large-scale substrates with highly aligned nanoscale grooves. Interestingly, although the cells strongly align with the nanoscale grooves, only a few also elongate along this axis: alignment of the cells does not require a pronounced change in morphology of the cell. We also pattern the nanotube bundles over length scales comparable to the cell size and show that the cells follow this pattern.

  18. Effects of drugs, x-rays, and heat on Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Tomasovic, S.P.

    1977-01-01

    The mitotic cell selection technique was used to monitor the effects of various drugs, primarily inhibitors of RNA synthesis, on x-ray-induced G2 delay. Addition of actinomycin D (2 μg/ml), caffeine (19--194 μg/ml), theophylline (18--180 μg/ml), or cordycepin (5--30 μg/ml) immediately before or after irradiation greatly reduced G2 delay and shifted the x-ray transition point (X-TP, the point in G2 beyond which cells are unaffected in their progression by x-rays) away from division. The magnitude of this protective effect increased with concentration. Addition of dimethylsulfoxide (10 6 μg/ml) immediately after irradiation reduced G2 delay but had no effect on the X-TP. The addition of 2-mercapto-1(β-4-pyridethyl) benzimidazole (25--75 μg/ml) or lucanthone (5--20 μg/ml) immediately before irradiation resulted in increased G2 delay, and shifted the X-TP closer to division. Studies of the effects of these drugs on incorporation of tritiated uridine or tritiated leucine into acid insoluble material indicated no correlation between reduction of G2 delay and rates of overall RNA or protein synthesis. Synchronous cells, treated continuously with 15 μg/ml of cordycepin starting in the latter part of S phase, proceeded into mitosis about 30 minutes ahead of controls. Howevr, cordycepin did not reduce mitotic delay observed for cells irradiated in S phase. Continuous treatment during G2 of unirradiated synchronous cells with 15 μg/ml of cordycepin had little effect on accelerating cells into mitosis, yet did reduce delay observed for cells irradiated in G2. These results are consistent with hypotheses requiring synthesis during G2 of critical protein molecules essential for mitosis. Heating mammalian cells induces lethality by an undetermined mechanism. Heat treatment of Chinese hamster ovary cells at 45.5 0 C resulted in an increase in nonhistone protein isolated with DNA

  19. Defective DNA cross-link removal in Chinese hamster cell mutants hypersensitive to bifunctional alkylating agents

    International Nuclear Information System (INIS)

    Hoy, C.A.; Thompson, L.H.; Mooney, C.L.; Salazar, E.P.

    1985-01-01

    DNA repair-deficient mutants from five genetic complementation groups isolated previously from Chinese hamster cells were assayed for survival after exposure to the bifunctional alkylating agents mitomycin C or diepoxybutane. Groups 1, 3, and 5 exhibited 1.6- to 3-fold hypersensitivity compared to the wild-type cells, whereas Groups 2 and 4 exhibited extraordinary hypersensitivity. Mutants from Groups 1 and 2 were exposed to 22 other bifunctional alkylating agents in a rapid assay that compared cytotoxicity of the mutants to the wild-type parental strain, AA8. With all but two of the compounds, the Group 2 mutant (UV4) was 15- to 60-fold more sensitive than AA8 or the Group 1 mutant (UV5). UV4 showed only 6-fold hypersensitivity to quinacrine mustard. Alkaline elution measurements showed that this compound produced few DNA interstrand cross-links but numerous strand breaks. Therefore, the extreme hypersensitivity of mutants from Groups 2 and 4 appeared specific for compounds the main cytotoxic lesions of which were DNA cross-links. Mutant UV5 was only 1- to 4-fold hypersensitive to all the compounds. Although the initial number of cross-links was similar for the three cell lines, the efficiency of removal of cross-links was lowest in UV4 and intermediate in UV5. These results suggest that the different levels of sensitivity are specifically related to different efficiencies of DNA cross-link removal. The phenotype of hypersensitivity to both UV radiation and cross-link damage exhibited by the mutants in Groups 2 and 4 appears to differ from those of the known human DNA repair syndromes

  20. Additive action of ionizing and non-ionizing radiations throughout the Chinese hamster cell-cycle

    International Nuclear Information System (INIS)

    Han, A.; Elkind, M.M.

    1977-01-01

    X-rays and γ-rays produce lesions in nuclear DNA which are qualitatively different from those produced by UV-light. Studies have been made of the effects of X-rays and UV light on the survival of synchronous cultures of Chinese hamster V79 cells. There were qualitative differences in the age-response patterns for survival after single doses of the two types of radiation, but combined UV-and X-irradiation produced enhanced lethality at all ages throughout the cell cycle. The minimum survival from the combined irradiation was at the middle of the S period, and the survival curves at this stage of the cell cycle were further investigated. Exposure to UV immediately before graded X-ray doses removed the shoulder on the X-ray survival curve in a progressive manner, while the D 0 value increased only slightly. The results correspond to complete additivity of X-ray damage to UV damage. Exposure to X-rays immediately before graded UV doses indicated that only part of the damage produced by the X-rays could be added to the UV-damage. Even after X-ray doses which reduced survival to levels which surpassed the shoulder of the UV-only survival curve, the shoulder persisted on the combined treatment survival curves. Measurements were made of the time-course of the change in molecular weight of single-stranded DNA after X-irradiation preceded by UV-irradiation. Only a small amount of slowing of repair of X-ray induced lesions was detected after a large UV dose. Possible mechanisms for the interactions between the two types of damage are discussed. (U.K.)

  1. Chinese hamster ovary cell mitosis and its response to ionizing radiation: A morphological analysis of the living cell

    International Nuclear Information System (INIS)

    Carlson, J.G.

    1989-01-01

    Repeated microscopic observations of exponentially growing Chinese hamster ovary cells were made and the times and mitotic stages were recorded in control and irradiated cultures at 37 degree C. As determined by autoradiography, the time from the end of S phase to early prophase (the G2 phase) was 46 min, to breakdown of the nuclear envelope was 91 min, and to restoration of the nuclear envelope was 116 min. The time spent in morphologically distinguishable phases of mitosis and the effects of 0.5, 1.0, 1.5, 2.0, and 4.0 Gy of gamma or X radiation on cells at each phase were determined. Affected cells were found to be delayed without or with reversion to an earlier mitotic stage before recovering and advancing through mitosis. Cells were timed in the five steps comprising delay with reversion: inertia, cessation I, regression, cessation II, and reprogression. No cells treated in late prophase, i.e., within 8-10 min of nuclear envelope breakdown, were delayed by the doses used; therefore the critical or transition point must be situated in middle prophase. Cells irradiated in this stage were not delayed by 0.5 or 1.0 Gy, but suffered a dose-dependent delay with or without reversion after 1.5, 2.0, and 4.0 Gy. Cells irradiated in early prophase and very late interphase responded similarly, but a greater percentage of the latter reverted

  2. Radiation effects on cultured mouse embryos in relation to cell division cycle

    International Nuclear Information System (INIS)

    Domon, M.

    1982-01-01

    The authors have worked with mouse embryos in vitro asking first, what are the suitable parameters to define the radiation sensitivity of embryos, and second what is a major factor determining it. The LD 50 was adopted as a parameter of the radiation sensitivity of a population in a mouse embryo system in culture. The fertilized ova were collected into Whitten's medium at various times during the pronuclear and 2-cell stages of development. They were irradiated in chambers with X-rays at doses of 0 to 800 rads. After the embryos were cultured, a set of the lethal fractions for various X-ray doses were obtained. Regarding the radiation sensitivity variation of the embryos, the LD 50 varied from 100 to 200 rads during the pronuclear stage and from 100 to 600 rads during the 2-cell stage. The embryos during the pronuclear stage were most radioresistant at early G 2 phase, followed by an increase in the sensitivity. The embryos during the 2-cell stage were also most radioresistant at early G 2 phase and were more sensitive when they got close to either the first or the second cleavage division. Furthermore, it seems that the factor 6 of the large variation was due to the extremely long G 2 period, 14 hrs for the 2-cell embryos. That is, the pooled 2-cell embryos were in a relative sense well synchronized with G 2 phase. In contrast, the synchrony was poor during the pronuclear stage, which led to less variation of the LD 50 for the pronuclear embryos. It is concluded that during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo. (Namekawa, K.)

  3. Change of nucleolus characteristic of fish embryo cells under the influence of low-level radiation

    International Nuclear Information System (INIS)

    Arkhipchuk, V.V.

    1995-01-01

    The nucleolus activity of fish embryo cells was stimulated by low-level radiation at a dose rate of 2-13 mGy/h. The size of nucleoli generally increased in embryos of Cyprinus carpio, whereas the number of nucleoli was greater in embryos of Carassius auratus gibelio. The higher the functional activity of nucleolus is, the more pronounced are changes in the characteristics. The size of single nucleolus at gastrulation is the most sensitive characteristic. 16 refs.; 1 tab

  4. New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

    Directory of Open Access Journals (Sweden)

    Kim Yeon-Gu

    2012-05-01

    Full Text Available Abstract Background The establishment of high producer is an important issue in Chinese hamster ovary (CHO cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. Results An internal ribosome entry site (IRES was introduced for using two green fluorescence protein (GFP fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (qAb than that of the unsorted pool. The qAb was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and qAb in individual selected clones. Conclusions This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of qAb with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

  5. Sensitization by wortmannin of heat- or X-ray induced cell death in cultured Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Tomita, Masanori; Suzuki, Norio; Matsumoto, Yoshihisa; Hirano, Kazuya; Umeda, Noriko; Sakai, Kazuo

    2000-01-01

    Here we found that wortmannin sensitized Chinese hamster V79 cells to hyperthermic treatment at 44.0 deg C as determined either by colony formation assay or by dye exclusion assay. Wortmannin enhanced heat-induced cell death accompanying cleavage of poly (ADP-ribose) polymerases (PARP). Additionally, the induction of heat shock protein HSP70 was suppressed and delayed in wortmannin-treated cells. Heat sensitizing effect of wortmannin was obvious at more than 5 or 10 μM of final concentrations, while radiosensitization was apparent at 5 μM. Requirement for high concentration of wortmannin, i.e., order of μM, suggests a possible role of certain protein kinases, such as DNA-PK and/or ATM among PI3-kinase family. The sensitization was minimal when wortmannin was added at the end of heat treatment. This was similar to the case of X-ray. Since heat-induced cell death and PARP cleavage preceded HSP70 induction phenomenon, the sensitization to the hyperthermic treatment was considered mainly caused by enhanced apoptotic cell death rather than secondary to suppression or delay by wortmannin of HSP70 induction. Further, in the present system radiosensitization by wortmannin was also at least partly mediated through enhancement of apoptotic cell death. (author)

  6. Effects of 1 MHz ultrasound on Chinese hamster V-79 cells: cavitational mechanisms and effects on proliferation

    International Nuclear Information System (INIS)

    Ciaravino, V.

    1982-01-01

    An assessment of acoustic cavitation as a primary physical mechanism in producing chemical and biological effects has been made. Chemical effects have been demonstrated through experimental protocols involving the release of iodine from sodium iodide. Biological effects have been shown by procedures assessing cell lysis and growth of in vitro Chinese hamsters V-79 cells. An important conclusion reached through these assessments is that the threshold level at which acoustic cavitation can exert an effect is dependent on the sensitivity of the experimental system being exposed. The proliferation of mitotically synchronous in vitro Chinese hamster V-79 cells exposed to 1 MHz ultrasound was investigated. Cell growth was assessed in the first three hours after sonication (3 W/cm 2 for 1 min) and was found to decrease to approx. 60 percent of control values. At an intensity of 3 W/cm 2 and exposure durations of 0.1, 1, 2, 5, and 10 min., mitotic cells underwent respectively increasing amounts of lysis. The remaining intact cells were observed for growth rate as indicated by the timed formation of colonies from single cells. The results indicated an immediate decrease in colony size (p 0.05)

  7. Protective action of DNA preparations on the survival of cells and yield of 8-azaguanine resistant mutations in X-irradiated cell culture of chinese hamsters

    International Nuclear Information System (INIS)

    Kuznetsova, N.N.; Feoktistova, T.P.

    1976-01-01

    A DNA preparation (molecular weight 19.6-21.0x1O 6 daltons) administered to cell culture of Chinese hamsters in concentrations of 100 to 122 μg/ml 60 minutes before and in the course of 3 days after X-irradiation (600 R) decreased the lethality of irradiated cells and reduced induction of 8-azaguanine resistant genic mutations. DNA preparations with the concentrations under study had no toxic action on cells and were not mutagenous

  8. Comparative study on fast neutrons radiobiological effect on Chinese hamster cells in culture depending on regime of irradiation

    International Nuclear Information System (INIS)

    Elisova, T.V.; Feoktistova, T.P.; Stavrakova, N.M.

    1988-01-01

    Comparative study of regularities of fast neutron radiobiological effect on Chinese hamster cells in culture under pulse and statistic irradiation regimes that was estimated by reproductive death of cells and induced frequency of resistence mutations to 6-tioguanine is carried out. It is stated that with the dose rate increase approximately by 6 orders radiobiological efficiency of fast neutrons decreases. It is suggested that one of the causes of decreasing pulse irradiation efficiency are processes on radiation-chemical level. 9 refs.; 3 figs

  9. Histochemical study of brown-fat cells in the golden hamster (Mesocricetus auratus) in cultures

    International Nuclear Information System (INIS)

    Sokolov, V.E.; Boyadzhieva-Mikhailova, A.; Koncheva, L.; Angelova, P.; Evgen'eva, T.P.

    1985-01-01

    The authors undertake the task of studying the synthesis of certain hormones by brown-fat cells. The authors used brown-fat cells from the golden hamster. The metabolism of brown-fat cells was studied on precultured cells, which made it possible to detect the synthesis of the studied substances rather than their accumulation in the organ. The authors conducted three experiments. First, fragments of brown fat were cultivated in diffusion chambers in vivo. Pieces of brown fat were cultivated in parallel in vitro on agar (organotypic cultures) and on plasma (histotypic cultures). During cultivation in diffusion chambers, the chambers were implanted in the abdominal cavity of young white rats. For in vitro cultivation, TCM 199 plus 15-20% calf serum was used. A total of 36 cultures with 12 cultures in each series of experiments were performed. The auto-radiographic studies of brown-fat cells were conducted on 24-hour cultures and on brown-fat fragments taken from the intact animal. The cultures were incubated with isotopes for 1 h. Either [ 3 H]lysine (87.3 Ci/mM specific activity), [ 3 H]arginine (16.7 Ci/mM), [ 3 H]glycerol (43 Ci/mM), or [ 3 H]cholesterol (43 Ci/mM) were added to the medium. After incubation, the cultures were washed three times in pure medium, fixed in Sierra fluid, and embedded in paraffin. The paraffin sections were covered with Ilford K 2 emulsion, and the preparations were exposed for 20 days at 4 0 C temperature. Radio-immunological methods were used to study the accumulation of estradiol-17-beta in the culture medium by the Dobson method and that of testerone. The culture medium was taken on cultivation days 2,4,6,8, and 10. The medium was changed during cultivation every third day, which made it possible to judge the rates of accumulation of material with increase in the cultivation times

  10. Chromosomal mosaicism in mouse two-cell embryos after paternal exposure to acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Marchetti, Francesco; Bishop, Jack; Lowe, Xiu; Wyrobek, Andrew J

    2008-10-14

    Chromosomal mosaicism in human preimplantation embryos is a common cause ofspontaneous abortions, however, our knowledge of its etiology is limited. We used multicolor fluorescence in situ hybridization (FISH) painting to investigate whether paternally-transmitted chromosomal aberrations result in mosaicism in mouse 2-cell embryos. Paternal exposure to acrylamide, an important industrial chemical also found in tobacco smoke and generated during the cooking process of starchy foods, produced significant increases in chromosomally defective 2-cell embryos, however, the effects were transient primarily affecting the postmeiotic stages of spermatogenesis. Comparisons with our previous study of zygotes demonstrated similar frequencies of chromosomally abnormal zygotes and 2-cell embryos suggesting that there was no apparent selection against numerical or structural chromosomal aberrations. However, the majority of affected 2-cell embryos were mosaics showing different chromosomal abnormalities in the two blastomeric metaphases. Analyses of chromosomal aberrations in zygotes and 2-cell embryos showed a tendency for loss of acentric fragments during the first mitotic division ofembryogenesis, while both dicentrics and translocations apparently underwent propersegregation. These results suggest that embryonic development can proceed up to the end of the second cell cycle of development in the presence of abnormal paternal chromosomes and that even dicentrics can persist through cell division. The high incidence of chromosomally mosaic 2-cell embryos suggests that the first mitotic division of embryogenesis is prone to missegregation errors and that paternally-transmitted chromosomal abnromalities increase the risk of missegregation leading to embryonic mosaicism.

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

    Directory of Open Access Journals (Sweden)

    Svetlana Gavrilov

    2011-01-01

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

  12. Nuclear and cellular expression data from the whole 16-cell stage Arabidopsis thaliana embryo and a cell type-specific expression atlas of the early Arabidopsis embryo

    NARCIS (Netherlands)

    Palovaara, J.P.J.

    2017-01-01

    SuperSeries contain expression data from the nuclei of cell types involved in patterning events, with focus on root apical stem cell formation, at 16-cell stage, early globular stage and late globular stage in the early Arabidopsis embryo (atlas). Expression data comparing nuclear and cellular RNA

  13. Using titer and titer normalized to confluence are complementary strategies for obtaining Chinese hamster ovary cell lines with high volumetric productivity of etanercept

    DEFF Research Database (Denmark)

    Pristovšek, Nuša; Hansen, Henning Gram; Sergeeva, Daria

    2018-01-01

    The selection of clonally-derived Chinese hamster ovary (CHO) cell lines with the highest production rate of recombinant glycoproteins remains a big challenge during early stages of cell line development. Different strategies using either product titer or product titer normalized to cell number...

  14. Size distribution of fullerenol nanoparticles in cell culture medium and their influence on antioxidative enzymes in Chinese hamster ovary cells

    Directory of Open Access Journals (Sweden)

    Srđenović Branislava U.

    2015-01-01

    Full Text Available Fullerenol (C60(OH24 nanoparticles (FNP have a significant role in biomedical research due to their numerous biological activities, some of which are cytoprotective and antioxidative properties. The aim of this study was to measure distribution of fullerenol nanoparticles and zeta potential in cell medium RPMI 1640 with 10% fetal bovine serum (FBS and to investigate the influence of FNP on Chinese hamster ovary cells (CHO-K1 survival, as well as to determine the activity of three antioxidative enzymes: superoxide-dismutase, glutathione-reductase and glutathione-S-transferase in mitomycin C-treated cell line. Our investigation implies that FNP, as a strong antioxidant, influence the cellular redox state and enzyme activities and thus may reduce cell proliferation, which confirms that FNP could be exploited for its use as a cytoprotective agent.[Projekat Ministarstva nauke Republike Srbije, br. III45005 i Pokrajinski Sekretarijat za nauku i tehnološki razvoj Vojvodine, grant number 114-451-2056/2011-01

  15. DNA repair in lens cells during chick embryo development

    International Nuclear Information System (INIS)

    Counis, M.F.; Chaudun, E.; Simonneau, L.; Courtois, Y.

    1979-01-01

    When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenon of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [ 3 H)thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in the experimental system the synthesis of delta- and αcrystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case. (Auth.)

  16. MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

    Directory of Open Access Journals (Sweden)

    STANCA CLAUDIA

    2007-01-01

    Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

  17. MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

    Directory of Open Access Journals (Sweden)

    CLAUDIA STANCA

    2007-05-01

    Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

  18. The increase in radioresistance of Chinese hamster cells cultured as spheroids is correlated to changes in nuclear morphology

    International Nuclear Information System (INIS)

    Gordon, D.J.; Milner, A.E.; Beaney, R.P.; Grdina, D.J.; Vaughan, A.T.

    1990-01-01

    Chinese hamster V79 cells grown as spheroids in roller culture are more radioresistant than those grown as monolayers. The supercoiled structure of chromatin, as salt-extracted nucleoids, has been examined using flow cytometry. Irradiated viable cells from spheroid culture contain restraints to supercoil relaxation that are absent in monolayer cells. Further analysis of the chromatin organization from each growth form shows that the radioresistant spheroid cells contain a DNA-protein matrix that is more resistant to detergent-induced degradation. The increase in structural integrity may be due to the retention of a 55-60 kDa protein that is apparent in the nucleoids of spheroid, but not monolayer cells. The increase in structural integrity of the spheroid cells may explain their greater radioresistance by providing a more stable platform for high-fidelity DNA damage repair

  19. Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

    Science.gov (United States)

    Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

    2015-11-01

    Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

  20. Efficiency of porcine somatic cell nuclear transfer – a retrospective study of factors related to embryo recipient and embryos transferred

    Directory of Open Access Journals (Sweden)

    Yongye Huang

    2013-10-01

    The successful generation of pigs via somatic cell nuclear transfer depends on reducing risk factors in several aspects. To provide an overview of some influencing factors related to embryo transfer, the follow-up data related to cloned pig production collected in our laboratory was examined. (i Spring showed a higher full-term pregnancy rate compared with winter (33.6% vs 18.6%, P = 0.006. Furthermore, a regression equation can be drawn between full-term pregnancy numbers and pregnancy numbers in different months (y = 0.692x−3.326. (ii There were no significant differences detected in the number of transferred embryos between surrogate sows exhibiting full-term development compared to those that did not. (iii Non-ovulating surrogate sows presented a higher percentage of full-term pregnancies compared with ovulating sows (32.0% vs 17.5%, P = 0.004; respectively. (iv Abortion was most likely to take place between Day 27 to Day 34. (v Based on Life Table Survival Analysis, delivery in normally fertilized and surrogate sows is expected to be completed before Day 117 or Day 125, respectively. Additionally, the length of pregnancy in surrogate sows was negatively correlated with the average litter size, which was not found for normally fertilized sows. In conclusion, performing embryo transfer in appropriate seasons, improving the quality of embryos transferred, optimizing the timing of embryo transfer, limiting the occurrence of abortion, combined with ameliorating the management of delivery, is expected to result in the harvest of a great number of surviving cloned piglets.

  1. Influence of irradiation at different stages of mitotic cycle upon production of sister chromatid exchanges in cultured Chinese hamster cells

    International Nuclear Information System (INIS)

    Antoshina, M.M.; Poryadkova, N.A.; Luchnik, N.V.

    1982-01-01

    Frequency of sister chromatid exchanges (SCE) and microexchanges in Chinese hamster cells has been studied by means of the method of differential staining of chromatids on irradiation at different stages of the mitotic cycle. It is shown that the irradiation enhances frequency of SCE and microexchanges if it is carried out before the end of DNA replication synthesis. Comparison of frequency depenedence of radiation-induced microexchanges and SCE at different stages of the mitotic cycle results in the conclusion that the microexchanges are none other than small SCE

  2. Alterations in body weight and blood glucose level of female hamsters exposed to electromagnetic fields of cell phones

    Directory of Open Access Journals (Sweden)

    A.R Lotfi

    2010-02-01

    Group 2 was exposed to electromagnetic field emitted by cell phones for 10 days (short term and group 3 for 50 day (long term. In the latter groups, the exposure was 1 hour per day. At the end of the experimental period, the animals were weighed and blood glucose concentrations were determined by obtaining blood samples from 8 randomly selected hamsters in each group.  The blood glucose level was significantly higher in long-term exposed group in comparison with the control and short-term exposed groups (175, 11.6 and 107 mg/dl, respectively (p

  3. Interaction of different forms of graphene with chicken embryo red blood cells

    DEFF Research Database (Denmark)

    Jaworski, S.; Hinzmann, Mateusz; Sawosz, Ewa

    2017-01-01

    , while others have indicated that graphene might become health hazards. In this study, we explore the biocompatibility of graphene-related materials with chicken embryo red blood cells (RBC). The hemolysis assay was employed to evaluate the in vitro blood compatibility of reduced graphene, graphene oxide......, and reduced graphene oxide, because these materials have recently been used for biomedical applications, including injectable graphene-related particles. This study investigated structural damage, ROS production and hemolysis of chicken embryo red blood cells. Different forms of graphene, when incubated...... with chicken embryo RBC, were harmful to cell structure and induced hemolysis....

  4. Histochemical, light and electron microscopic study of polonium-210 induced peripheral tumours in hamster lungs -evidence implicating the Clara Cell as the cell of origin

    International Nuclear Information System (INIS)

    Kennedy, A.R.; McGandy, R.B.; Little, J.B.

    1977-01-01

    Peripheral lung tumors induced in Syrian golden hamsters by intratracheally administered polonium-210 ( 210 Po) are similar to the peripheral lung tumours induced in many species by a variety of carcinogens. In addition, they show many of the histopathological features observed in human bronchiolar-alveolar carcinomas. Serial sacrifice studies of hamsters exposed to multiple instillations of 210 Po have been carried our to identify the cell of origin of these tumors. By means of thin, plastic (glycol methacrylate) sections, electron microscopy, and histochemistry, it is concluded that the bronchiolar Clara cell is the probable cell of origin, and that this view is generally compatible with many of the reported cytological characteristics of the human tumor. (author)

  5. Derivation of Rabbit Embryonic Stem Cells from Vitrified–Thawed Embryos

    Science.gov (United States)

    Chen, Chien-Hong; Li, Yi; Hu, Yeshu; An, Li-You; Yang, Lan; Zhang, Jifeng; Chen, Y. Eugene

    2015-01-01

    Abstract The rabbit is a useful animal model for regenerative medicine. We previously developed pluripotent rabbit embryonic stem cell (rbESC) lines using fresh embryos. We also successfully cryopreserved rabbit embryos by vitrification. In the present work, we combined these two technologies to derive rbESCs using vitrified–thawed (V/T) embryos. We demonstrate that V/T blastocysts (BLs) can be used to derive pluripotent rbESCs with efficiencies comparable to those using fresh BLs. These ESCs are undistinguishable from the ones derived from fresh embryos. We tested the developmental capacity of rbESCs derived from V/T embryos by BL injection experiments and produced chimeric kits. Our work adds cryopreservation to the toolbox of rabbit stem cell research and applications and will greatly expand the available research materials for regenerative medicine in a clinically relevant animal model. PMID:26579970

  6. Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Matzuk, M.M.; Krieger, M.; Corless, C.L.; Boime, I.

    1987-01-01

    Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common α subunit but differ in their hormone-specific β-subunits. The β subunit of hCG (hCGβ) is unique among the β subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCGβ gene alone or together with the hCGα gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results reveal that hCGβ can be secreted normally in the absence of its O-linked oligosaccharides. hCGβ devoid of O-linked carbohydrate can also combine efficiently with hCGα and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCGβ O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG

  7. The neuroblast of the grasshopper embryo as a new mutagen test system. Pt. 1

    International Nuclear Information System (INIS)

    Liang, J.C.; Gaulden, M.E.

    1982-01-01

    The neuroblasts of the grasshopper embryo (Chortophaga viridifasciata De Geer) are being studied to determine their suitability for detecting environmental clastogens (chromosome-breaking agents). They are very sensitive to the induction of chromosome breakage by radiation in viro. Their sensitvity, 0.011 break/cell/R, is 4-5 times higher than pollen mother cells of Tradescantia (micronuclei), 10 times higher than either human lymphocytes or Chinese hamster cells (metaphase chromosome aberrations), and 15 times higher than mouse erythroblasts (micronuclei). Furthermore, they have no spontaneous chromosome breakage, which facilitates the detection of agents that break chromosomes. The present study shows that Chortophaga embryos maintain normal mitotic activity in vitro for 5 cell cycles at 38 0 C (20 h), and that neuroblasts of embryos grown in vitro have the same radiosensitivity as those of embryos in vivo. Thus in vitro exposure of grasshopper embryos is a promising method for obtaining data on the response of neuroblasts to chemical clastogens. (orig.)

  8. Neuroblast of the grasshopper embryo as a new mutagen test system. Pt. 1. In vitro radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Liang, J C; Gaulden, M E [Texas Univ., Dallas (USA). Dept. of Radiology

    1982-04-01

    The neuroblasts of the grasshopper embryo (Chortophaga viridifasciata De Geer) are being studied to determine their suitability for detecting environmental clastogens (chromosome-breaking agents). They are very sensitive to the induction of chromosome breakage by radiation in vitro. Their sensitvity, 0.011 break/cell/R, is 4-5 times higher than pollen mother cells of Tradescantia (micronuclei), 10 times higher than either human lymphocytes or Chinese hamster cells (metaphase chromosome aberrations), and 15 times higher than mouse erythroblasts (micronuclei). Furthermore, they have no spontaneous chromosome breakage, which facilitates the detection of agents that break chromosomes. The present study shows that Chortophaga embryos maintain normal mitotic activity in vitro for 5 cell cycles at 38/sup 0/C (20 h), and that neuroblasts of embryos grown in vitro have the same radiosensitivity as those of embryos in vivo. Thus in vitro exposure of grasshopper embryos is a promising method for obtaining data on the response of neuroblasts to chemical clastogens.

  9. Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Kaina, B.; Van Zeeland, A.A.; Backendorf, C.; Thielmann, H.W.; Van de Putte, P.

    1987-01-01

    Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA

  10. Gene mutations, chromosome aberrations and survival after X-ray irradiation of cultured Chinese hamster cells at cysteamine protection

    International Nuclear Information System (INIS)

    Elisova, I.V.; Feoktistova, I.P.

    1983-01-01

    The culture of Chinese hamster cells (clone 431) has been used to study cysteamine action on mutagenous effect of X-rays, determined by the induction of resistance of gene mutations to 6-thioguanine and chromosomal abberations, as well as on the reproductive form of death of irradiated cells. Dose--- effect curves are obtained under conditions of irradiation with and without protector. The factor of dose alteration is 2.0 for chromosomal aberrations and cell survival, and 2.8 for gene mutations. It is sUpposed that cysteamine affects the general mechanisms, which take part in the realis zation of injuries that bring about gene mutations, chromosomal aberrations and cell lethality

  11. Cyclical and patch-like GDNF distribution along the basal surface of Sertoli cells in mouse and hamster testes.

    Directory of Open Access Journals (Sweden)

    Takeshi Sato

    Full Text Available BACKGROUND AND AIMS: In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs through GDNF family receptor α1 (GFRα1. It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. CONCLUSION/SIGNIFICANCE: Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules.

  12. Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

    Science.gov (United States)

    Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

    2015-04-01

    Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Anomalous dose-response characteristics induced by caffeine in ultraviolet-irradiated V79-79 Chinese hamster cells

    International Nuclear Information System (INIS)

    Schroy, C.B.; Todd, P.

    1979-01-01

    Cultured Chinese hamster cell line V79-79 exhibited an increase in survival with increasing UV fluence after a sharp decrease when exposed to 2.5 mM caffeine for 44 h after far-UV irradiation resulting in an anomalous maximum in the survival curve. No survival maximum was evident when either 0 or 1 mM caffeine is administered under the same conditions. The UV survival curve for 2.5 mM caffeine crossed the corresponding 1 mM curve and apparently became asymptotic to the 0 mM curve as UV fluence was increased. Chinese hamster cell lines V79-753B (related to V79-79 by derivation from the same parental line) and M3-1F3 (unrelated) exhibited only potentiation of post-UV lethality by the same concentration of caffeine and had no caffeine-induced anomalies in their survival curves. Xanthine, used alone or in combination with caffeine, only potentiated a slight amount of lethality and appeared not to be a major causative factor of the anomaly. (author)

  14. Modification of UV-induced mutation frequencies in Chinese hamster- cells by dose fractionation, cycloheximide and caffeine treatments

    International Nuclear Information System (INIS)

    Chang, C.-C.; Schultz, R.; Trosko, J.E.; D'Ambrosio, S.M.; Setlow, R.B.

    1978-01-01

    Chinese hamster (V79) cells were irradiated with a fractionated regime of ultraviolet light (UV 1 +UV 2 ). The fractionation of a UV dose always increased the colony-forming ability but reduced (or it did not change) the mutation frequencies. Treatment with cycloheximide between the two UV irradiations resulted in two types of effects, depending on the protocols used. Long exposures to cycloheximide (i.e., >6h) for the entire period between UV 1 and UV 2 or partial treatment of cycloheximide (i.e., 3h) long before UV 2 always resulted in reduced colony-forming ability and enhanced or unchanged mutation frequencies. Exposure to cycloheximide for the entire period in the short fractionated regime (i.e., 4h) between UV 1 and UV 2 or partial treatment of cycloheximide just prior to UV 2 tended to give the opposite effects. Caffeine treatment before UV 2 , with or without UV 1 , significantly increased the mutation frequencies. These results suggest that an error-free postreplication repair system exists in Chinese hamster cells which is inhibitable by particular cycloheximide or caffeine treatments. (Auth.)

  15. Viability test of fish scale collagen (Oshpronemus gouramy on baby hamster kidney fibroblasts-21 fibroblast cell culture

    Directory of Open Access Journals (Sweden)

    Chiquita Prahasanti

    2018-04-01

    Full Text Available Aim: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy by evaluating its viability against baby hamster kidney fibroblasts-21. Materials and Methods: Collagen was extracted from gouramy fish scales (O. gouramy with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. Results: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed value of 0.754 (p>00025. Conclusion: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

  16. Studies into the protein content in hamster kidney cells BHK21 infected with the fowl plague virus

    International Nuclear Information System (INIS)

    Gagov, I.I.; Trifonov, S.D.; Aleksandrov, I.I.

    1977-01-01

    The impact of viral infection on protein synthesis has been studied in vitro. Cultures using a stable cell line, BHK 21 (hamster kidney cells), are inoculated with chicken-pox virus and scores are made of 14 C-lysine incorporation rate. The results indicate depression of protein synthesis, particularly marked within the first few hours after inoculation; at 5 hours the level of inrorporation into water soluble proteins is as low as about 35% of the control level, by 24 hours it is only about 19%. Distribution among 13 electrophoretic fractions of water soluble proteins is found to vary over a wide range, mobile fractions showing higher rates of amino acid incorporation while others exhibit depression or figures of the same order of magnitude as controls. It may thus be inferred that, in cell cultures, virus inoculation preferentially affects only some portion of protein synthesis. (A.B.)

  17. The suppressive effect of etoposide on recovery from sublethal radiation damage in Chinese hamster V 79 cells

    International Nuclear Information System (INIS)

    Saito, Tsutomu; Shimada, Yuji; Kawamori, Jiro; Kamata, Rikisaburo

    1992-01-01

    The combined effect of radiation and etoposide on the survival of cultured Chinese hamster V 79 cells was investigated. Cells in exponential growth phase were treated with various combinations of radiation and etoposide. The surviving fraction was assessed by colony formation. Etoposide significantly reduced so-called shoulder width, as expressed in Dq (quasithreshold dose), of radiation survival curves. The reduction depended on the increase of etoposide concentrations, although steepening of slopes of exponentially regressing portions of the radiation survival curves was slight. Split dose experiments showed that cells did not recover from sublethal radiation damage in the presence of low concentration of etoposide, although they did recover from sublethal radiation damage under a drug free condition. The results show the suppressive effect of etoposide on recovery from sublethal radiation damage. The effect of a sequential combination of radiation and etoposide was also investigated. The effect was more marked when the interval between radiation and etoposide was shorter regardless of the sequence. (author)

  18. Influence of DMSO on Carbon K ultrasoft X-rays induced chromosome aberrations in V79 Chinese hamster cells

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, Adayapalam T., E-mail: natarajan@live.nl [University of Tuscia, Viterbo (Italy); Palitti, Fabrizio [University of Tuscia, Viterbo (Italy); Hill, Mark A. [CRUK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ (United Kingdom); MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 0RD (United Kingdom); Stevens, David L. [MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire OX11 0RD (United Kingdom); Ahnstroem, Gunnar [Department of Microbiology and Genetic Toxicology, Stockholm University, Stockholm (Sweden)

    2010-09-10

    Ultrasoft X-rays have been shown to be very efficient in inducing chromosomal aberrations in mammalian cells. The present study was aimed to evaluate the modifying effects of DMSO (a potent scavenger of free radicals) on the frequencies of chromosome aberrations induced by soft X-rays. Confluent held G1 Chinese hamster cells (V79) were irradiated with Carbon K ultrasoft X-rays in the presence and absence of 1 M DMSO and frequencies of chromosome aberrations in the first division cells were determined. DMSO reduced the frequencies of exchange types of aberrations (dicentrics and centric rings) by a factor of 2.1-3.5. The results indicate that free radicals induced by ultrasoft X-rays contribute to a great extent to the induction of chromosome aberrations. The possible implications of these results in interpreting the mechanisms involved in the high efficiency of ultrasoft X-rays in the induction of chromosome aberrations are discussed.

  19. Lack of specificity of chromosome breaks resulting from radiation-induced genomic instability in Chinese hamster cells

    International Nuclear Information System (INIS)

    Trott, K.-R.; Teibe, A.

    1998-01-01

    In V79 Chinese hamster cells, radiation-induced genomic instability results in a persistently increased frequency of micronuclei, dicentric chromosomes and apoptosis and in decreased colony-forming ability. These manifestations of radiation-induced genomic instability may be attributed to an increased rate of chromosome breakage events many generations after irradiation. This chromosomal instability does not seem to be a property which has been inflicted on individual chromosomes at the time of irradiation. Rather, it appears to be secondary to an increased level of non-specific clastogenic factors in the progeny of most if not all irradiated cells. This conclusion is drawn from the observations presented here, that all the chromosomes in surviving V79 cells are involved in the formation of dicentric chromosome aberrations 1 or 2 weeks after irradiation with about equal probability if corrections are made for chromosome length. (orig.)

  20. Fibroblast receptor for cell-substratum adhesion: studies on the interaction of baby hamster kidney cells with latex beads coated by cold insoluble globulin (plasma fibronectin)

    OpenAIRE

    1980-01-01

    Studies were carried out on the interactions of uncharged latex beads (0.76 micrometer) with baby hamster kidney cells. Binding of beads to the cells occurred if the beads were coated by cold insoluble globulin (CIG) (plasma fibronectin) but not if the beads were coated by bovine albumin. Bovine albumin-coated beads did not bind to the cells even in the presence of excess CIG in the incubation medium. Binding of beads occurred randomly over the entire surfaces of cells in suspension. However,...

  1. Evaluation of cell number and DNA content in mouse embryos cultivated with uranium

    International Nuclear Information System (INIS)

    Kundt, Mirian S.; Cabrini, Romulo L.

    2000-01-01

    The evaluation of the degree of development, the number of cells and the DNA content, were used to evaluate the embryotoxicity of uranium. Embryos at a one cell stage were cultured with uranyl nitrate hexahydrate (UN) at a final concentration of uranium (U) of 26, 52 and 104 μgU/ml. At 24 hs of culture, the embryos at the 2 cell stage, were put in new wells with the same concentrations of U as the previous day, until the end of the period of incubation at 72 hs. At 72 hs of culture, 87% of the original one cell embryos were at morula stage, and in those cultivated with uranium, the percentage decreased significantly to 77; 63.24 and 40.79% respectively for the different U concentrations. Those embryos that exhibited a normal morphology, were selected and fixed on slides. The number of cells per embryo was evaluated in Giemsa stained preparations. The DNA content was evaluated cytophotometrically in Feulgen stained nuclei. The number of cells decreased significantly from 20,3 ± 5.6 in the control to 19 ± 6; 14 ± 3 and 13.9 ± 5.6 for the different concentrations. All the embryos evaluated showed one easy recognizable polar body, which was used a haploid indicator (n). The content of DNA was measured in a total of 20 control embryos and 16 embryos cultivated with UN. In control embryos, 92,7% of the nuclei presented a normal ploidy from 2n to 4n, 2,9% nuclei were hypoploid and 4,4% were hyperploid. The percentage of hypoploid nuclei rose in a dose-dependent fashion to 3.45; 44.45 and 50.34% respectively for the embryos cultured at the different U concentrations. The results indicate that U is embryotoxic, that its effects are dose dependent at the concentrations used in this study and that even those embryos that show a normal morphology, can be genetically affected. We show that the model employed is extremely sensitive. It is possible to use the preimplantation embryos, as a model to test the effect of possibly mutagenic agents of the nuclear industry. (author)

  2. Ultra-fast repair of single-strand breaks in DNA of. gamma. -irradiated Chinese hamster cells

    Energy Technology Data Exchange (ETDEWEB)

    Leontjeva, G A; Mantzighin, Yu A; Gaziev, A I [AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki

    1976-12-01

    Studies of the effect of thermal treatment of Chinese hamster cells on sedimentation of DNA in the alkaline sucrose gradient showed that heating the cells to 68/sup 0/C for 15 min caused the same degradation as ..gamma..-irradiation with 5 to 7 krad at 37/sup 0/C. The inhibition of cellular repair enzymes by heating was therefore unacceptable. The process of ultra-fast repair is essentially determined by the DNA-ligase reaction, which is activated in the presence of Mg ions, and inhibited in mammalian cells in the presence of EDTA and pyrophosphate. Sedimentation profiles were therefore measured for the DNA of Chinese hamster cells ..gamma..-irradiated (5 krad) at 0/sup 0/C or 22/sup 0/C in the presence of Mg/sup + +/, or EDTA and pyrophosphate, and the results demonstrated ultra-fast repair only at 20 to 37/sup 0/C, in contrast to bacteria. A study was made of the temperature dependence of the activity of the DNA ligases isolated from E.coli and rabbit bone marrow. The NAD-dependent bacterial DNA ligase was active at temperatures from 0 to 40/sup 0/C, whereas ATP-dependent DNA ligase of mammals only showed activity in the range 15 to 40/sup 0/C. The differing temperature dependences of ultra-fast repair in bacterial and mammalian cells are in agreement with the temperature dependences of the activities of isolated enzymes, and the results suggest that the process of ultra-fast repair of single-strand breaks of DNA takes place in both bacterial and mammalian cells.

  3. Transfer of Chinese hamster DNA repair gene(s) into repair-deficient human cells (Xeroderma pigmentosum)

    International Nuclear Information System (INIS)

    Karentz, D.; Cleaver, J.E.

    1985-01-01

    Transfer of repair genes by DNA transfection into repair-deficient Xeroderma pigmentosum (XP) cells has thus far been unsuccessful, presenting an obstacle to cloning XP genes. The authors chose an indirect route to transfer repair genes in chromosome fragments. DNA repair-competent (UV resistant) hybrid cell lines were established by PEG-mediated fusions of DNA repair-deficient (UV sensitive) human fibroblasts (XP12RO) with wild type Chinese hamster (CHO) cells (AA8). CHO cells were exposed to 5 Krad X-rays prior to fusions, predisposing hybrid cells to lose CHO chromosome fragments preferentially. Repair-competent hybrids were selected by periodic exposures to UV light. Secondary and tertiary hybrid cell lines were developed by fusion of X-irradiated hybrids to XP12RO. The hybrid cell lines exhibit resistance to UV that is comparable to that of CHO cells and they are proficient at repair replication after UV exposure. Whole cell DNA-DNA hybridizations indicate that the hybrids have greater homology to CHO DNA than is evident between XP12RO and CHO. These observations indicate that CHO DNA sequences which can function in repair of UV-damaged DNA in human cells have been transferred into the genome of the repair-deficient XP12RO cells

  4. Enhancement of excision-repair efficiency by conditioned medium from density-inhibited cultures in V79 Chinese hamster cells

    International Nuclear Information System (INIS)

    Nakano, S.

    1979-01-01

    Conditioned medium from density-inhibited V79 Chinese hamster cell cultures, given as a post-treatment to UV-irradiated homologous cells, was demonstrated to reduce the lethal action of ultraviolet light by temporarily blocking DNA replication. Since the increased survival was not affected by various nontoxic concentrations of caffeine, such protective effect would be attributable to the prolonged intervention of excision repair before DNA replication during the post-treatment period. The influence of conditioned medium on the UV-induced mutation at the ouabain-resistance locus was also examined and a significant decrease in mutation frequecy was noted. The observed reduction in killing and mutation as a result of post-incubation in conditioned medium, which delays DNA replication, would be interpreted as evidence that conditioned medium provides a longer period of time for an error-free excision-repair process, leaving lesion in DNA available for error-prone post-replication repair. (Auth.)

  5. Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Cirullo, R.E.; Dana, S.; Wasmuth, J.J.

    1983-01-01

    A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure

  6. Effect of secondary radiation from 70 GeV protons and γ-quanta on Chinese hamster chromosomes depending on the cell cycle stage

    International Nuclear Information System (INIS)

    Antipov, A.V.; Aptikaeva, G.F.; Akhmadieva, A.Kh.; Ganassi, E.Eh.; Zaichkina, S.I.; Livanova, I.A.; Smirnova, E.N.

    1987-01-01

    In cultured Chinese hamster cells, no decrease in the number of chromosome aberrations was noted after exposure thereof to 70 GeV protons at the late S-phase as opposed to early one. It is suggested that high biological effectiveness of this type of raiation is associated with its inhibiting effect of cytogenetic damages repair

  7. A physiological threshold for protection against menadione toxicity by human NAD(P)H : quinone oxidoreductase (NQO1) in Chinese hamster ovary (CHO) cells

    NARCIS (Netherlands)

    Haan, de L.H.J.; Boerboom, A.M.J.F.; Rietjens, I.M.C.M.; Capelle, van D.; Ruijter, de A.J.M.; Jaiswal, A.K.; Aarts, J.M.M.J.G.

    2002-01-01

    NAD(P)H:quinone oxidoreductase 1 (NQO1) has often been suggested to be involved in cancer prevention by means of detoxification of electrophilic quinones. In the present study, a series of Chinese hamster ovary (CHO) cell lines expressing various elevated levels of human NQO1 were generated by

  8. Accelerated Homology-Directed Targeted Integration of Transgenes in Chinese Hamster Ovary Cells Via CRISPR/Cas9 and Fluorescent Enrichment

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Grav, Lise Marie; Pedersen, Lasse Ebdrup

    2016-01-01

    Targeted gene integration into site-specific loci can be achieved in Chinese hamster ovary (CHO) cells via CRISPR/Cas9 genome editing technology and the homology-directed repair (HDR) pathway. The low efficiency of HDR often requires antibiotic selection, which limits targeted integration...

  9. Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Hård, K.; Mekking, A.; Damm, J.B.L.; Kamerling, J.P.; Boer, W. de; Wijnands, R.A.

    1990-01-01

    Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides were separated from

  10. Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. I. A cell proliferation study

    NARCIS (Netherlands)

    Rutten, A.A.J.J.L.; Wilmer, J.W.G.M.; Beems, R.B.

    1988-01-01

    The effects of cigarette smoke condensate (CSC) and all-trans retinol on the cell proliferative activity of vitamin A-deprived hamster tracheal epithelium have been studied in vitamin A-deficient, serum-free, hormone-supplemented medium in organ culture. In the absence of retinol, CSC induced a

  11. The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo.

    Science.gov (United States)

    Capmany, G; Taylor, A; Braude, P R; Bolton, V N

    1996-05-01

    The timing of pronuclear formation and breakdown, DNA synthesis and cleavage during the first cell cycle of human embryogenesis are described. Pronuclei formed between 3 and 10 h post-insemination (hpi; median 8 hpi). S-phase commenced between 8 and 14 hpi, and was completed between 10 and 18 hpi. M-phase was observed between 22 and 31 hpi (median duration 3 h), and cleavage to the 2-cell stage took place between 25 and 33 hpi. The timing of the same events was determined in 1-cell embryos derived from re-inseminated human oocytes that had failed to fertilize during therapeutic in-vitro fertilization (IVF). In these embryos, pronuclei formed between 3 and 8 h post-re-insemination (hpr-i), coinciding with the beginning of S-phase. While S-phase was completed as early as 10 hpr-i in some embryos, it extended until at least 16 hpr-i in others. Pronuclear breakdown and cleavage occurred from 23 and 26 hpr-i respectively; however, they did not occur in some embryos until after 46 hpr-i. The results demonstrate a markedly greater degree of variation in the timing of these events in embryos derived from re-inseminated oocytes compared with embryos derived from conventional IVF, and thus throw into question the validity of using the former as models for studies of the first cell cycle of human embryogenesis.

  12. Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

    International Nuclear Information System (INIS)

    Verma, S.P.; Sonwalkar, N.

    1991-01-01

    The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between -10 and 5 degree C (low-temperature transition), 10 and 22 degree C (middle-temperature transition), and 32 and 40 degree C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14 degree C). Second, the middle-temperature transition shifts up to the range of about 20-32 degree C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of about 15-40 degree C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties

  13. Cell membrane and cell junctions in differentiation of preimplanted mouse embryos.

    Science.gov (United States)

    Izquierdo, L; Fernández, S; López, T

    1976-12-01

    Cell membrane and cell junctions in differentiation of preimplanted mouse embryos, (membrana celular y uniones celulares en la diferenciación del embrión de ratón antes de la implantación). Arch. Biol. Med. Exper. 10: 130-134, 1976. The development of cell junctions that seal the peripheral blastomeres could be a decisive step in the differentiation of morulae into blastocysts. The appearance of these junctions is studied by electron microscopy of late morulae and initial blastocysts. Zonulae occludentes as well as impermeability to lanthanum emulsion precedes the appearance of the blastocel and hence might be considered as one of its necessary causes.

  14. X-ray induced changes in immunostaining of proliferating cell nuclear antigen (PCNA) in V79 hamster fibroplasts

    International Nuclear Information System (INIS)

    Lohr, F.; Hof, H.; Weber, K.-J.; Latz, D.; Wenz, F.

    1998-01-01

    To better understand the role of PCNA after photon irradiation in vivo, using flow cytometry, we studied the immunochemical PCNA-staining in V79 cells after irradiation with 6-MeV photons with and without serum depletion and with and without low-dose pre-irradiation under different growth conditions. Material and methods: Using V79 hamster cells, BrdUrd incorporation, total and DNA-bound PCNA were measured for exponential cells and for confluent cells at different times (up to 14 days) after reaching confluence. Cells were either grown with medium containing 10% fetal calf serum (FCS) or 0.5% FCS. Six days after reaching confluence, cells were irradiated with 1 Gy (and 8 Gy for non-serum-depleted cells) (6-MV photons, 2 Gy/min). Then, immunochemical PCNA-staining was measured by flow cytometry at 0, 30, 60 and 120 min after irradiation. For studying the adaptive response, exponentially growing cells and cells that were 6 days in confluence were pretreated with 0.01 Gy, reincubated for 5 h and then definitively treated with 1 Gy and harvested and processed as described above. Results: Four days after reaching confluence, DNA-bound PCNA and BrdUrd content were reduced to a minimum of [de

  15. Gene expression of bovine embryos developing at the air-liquid interface on oviductal epithelial cells (ALI-BOEC).

    Science.gov (United States)

    van der Weijden, Vera A; Chen, Shuai; Bauersachs, Stefan; Ulbrich, Susanne E; Schoen, Jennifer

    2017-11-25

    We recently developed an air-liquid interface long-term culture of differentiated bovine oviductal epithelial cells (ALI-BOEC). This ex vivo oviduct epithelium is capable of supporting embryo development in co-culture up to the blastocyst stage without addition of embryo culture medium. However, blastocyst rates in co-culture were markedly lower than in conventional in vitro embryo production procedures. In the present study, we assessed target gene expression of ALI-BOEC derived embryos to test their similarity to embryos from conventional in vitro embryo culture. We screened previously published data from developing bovine embryos and selected 41 genes which are either differentially expressed during embryo development, or reflect differences between various in vitro culture conditions or in vitro and in vivo embryos. Target gene expression was measured in 8-cell embryos and blastocysts using a 48.48 Dynamic Array™ on a Biomark HD instrument. For comparison with the ALI-BOEC system, we generated embryos by two different standard IVP protocols. The culture conditions lead to differential gene expression in both 8-cell embryos and blastocysts. Across the expression of all target genes the embryos developing on ALI-BOEC did not depart from conventional IVP embryos. These first results prove that gene expression in ALI-BOEC embryos is not largely aberrant. However, there was no clear indication for a more in vivo-like target gene expression of these embryos. This calls for further optimization of the ALI-BOEC system to increase its efficiency both quantitatively and qualitatively.

  16. Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

    Science.gov (United States)

    Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

    2013-05-01

    This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

  17. An integrated modelling framework from cells to organism based on a cohort of digital embryos

    OpenAIRE

    Villoutreix, Paul; Delile, Julien; Rizzi, Barbara; Duloquin, Louise; Savy, Thierry; Bourgine, Paul; Doursat, Ren?; Peyri?ras, Nadine

    2016-01-01

    We conducted a quantitative comparison of developing sea urchin embryos based on the analysis of five digital specimens obtained by automatic processing of in toto 3D+ time image data. These measurements served the reconstruction of a prototypical cell lineage tree able to predict the spatiotemporal cellular organisation of a normal sea urchin blastula. The reconstruction was achieved by designing and tuning a multi-level probabilistic model that reproduced embryo-level dynamics from a small ...

  18. High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Esther K. Kieserman, Chanjae Lee, Ryan S. Gray, Tae Joo Park and John B. Wallingford Corresponding author ([]()). ### INTRODUCTION Embryos of the frog *Xenopus laevis* are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than in...

  19. Stem cells from residual IVF-embryos - Continuation of life justifies isolation.

    NARCIS (Netherlands)

    Bongaerts, G.P.A.; Severijnen, R.S.V.M.

    2007-01-01

    Embryonic stem cells are undifferentiated pluripotent cells that can indefinitely grow in vitro. They are derived from the inner mass of early embryos. Because of their ability to differentiate into all three embryonic germ layers, and finally into specialized somatic cell types, human embryonic

  20. Hamster-Adapted Sin Nombre Virus Causes Disseminated Infection and Efficiently Replicates in Pulmonary Endothelial Cells without Signs of Disease

    OpenAIRE

    Safronetz, David; Prescott, Joseph; Haddock, Elaine; Scott, Dana P.; Feldmann, Heinz; Ebihara, Hideki

    2013-01-01

    To date, a laboratory animal model for the study of Sin Nombre virus (SNV) infection or associated disease has not been described. Unlike infection with Andes virus, which causes lethal hantavirus pulmonary syndrome (HPS)-like disease in hamsters, SNV infection is short-lived, with no viremia and little dissemination. Here we investigated the effect of passaging SNV in hamsters. We found that a host-adapted SNV achieves prolonged and disseminated infection in hamsters, including efficient rep...

  1. Amino acid sequence and posttranslational modifications of human factor VIIa from plasma and transfected baby hamster kidney cells

    International Nuclear Information System (INIS)

    Thim, L.; Bjoern, S.; Christensen, M.; Nicolaisen, E.M.; Lund-Hansen, T.; Pedersen, A.H.; Hedner, U.

    1988-01-01

    Blood coagulation factor VII is a vitamin K dependent glycoprotein which in its activated form, factor VII a , participates in the coagulation process by activating factor X and/or factor IX in the presence of Ca 2+ and tissue factor. Three types of potential posttranslational modifications exist in the human factor VII a molecule, namely, 10 γ-carboxylated, N-terminally located glutamic acid residues, 1 β-hydroxylated aspartic acid residue, and 2 N-glycosylated asparagine residues. In the present study, the amino acid sequence and posttranslational modifications of recombinant factor VII a as purified from the culture medium of a transfected baby hamster kidney cell line have been compared to human plasma factor VII a . By use of HPLC, amino acid analysis, peptide mapping, and automated Edman degradation, the protein backbone of recombinant factor VII a was found to be identical with human factor VII a . Asparagine residues 145 and 322 were found to be fully N-glycosylated in human plasma factor VII a . In the recombinant factor VII a , asparagine residue 322 was fully glycosylated whereas asparagine residue 145 was only partially (approximately 66%) glycosylated. Besides minor differences in the sialic acid and fucose contents, the overall carbohydrate compositions were nearly identical in recombinant factor VII a and human plasma factor VII a . These results show that factor VII a as produced in the transfected baby hamster kidney cells is very similar to human plasma factor VII a and that this cell line thus might represent an alternative source for human factor VII a

  2. MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

    Science.gov (United States)

    Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

    2011-02-15

    At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

  3. [INFLUENCE OF NANODIAMONDS AND CARBON NANOWIRES ON SURVIVAL AND CELLS STRUCTURE IN CHICKEN EMBRYO].

    Science.gov (United States)

    Lavrinenko, V; Zinabadinova, S; Chaikovsky, Yu; Sokurenko, L; Shobat, L

    2016-06-01

    Aim - to determine the effect of nanodiamonds and carbon nanowires on the survival and ultrastructure of chicken embryo cells. The experiment was carried out on chicken embryos, incubated from eggs of Hy-Line breed. Control and two experimental groups were formed (total number of embryos - 100). Diamond nanoparticles and carbon nanowires were administered on day 3 of incubation as a suspension of a biocompatible dextran. Ultrastructural analysis and general study of embryos state were carried out. The most expressed pathological effects were observed in the group with the introduction of the CNW, which caused visual impairment of embryogenesis that started from the early incubation periods. As for ND we can claim their prolonged impact on the development of embryos, manifested in the gradual deterioration of the embryos condition with the manifestations of the pathology in the provisory organs and the body of embryos. The results of our study demonstrate that both types of nanostructures can cause sublethal and irreversible morphologic changes. Detection of morphological evidence of the impact of nanomaterials at significant distances from the site of administration of nanoparticles shows highly penetrating ability of nanomaterials. The presence of damages specific for each type of nanoparticles shows affinity to various tissues and cellular structures. It is demonstrated that similar, at first glance, impact of nanomaterials, such as the induction of oxidative stress might be caused by specific structural transformations. So, ND cause vacuolization of mitochondria, and the CNW - deformation of their shape and appearance of dark inclusions in them.

  4. Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

    Science.gov (United States)

    Ono, Yukiko; Kono, Tomohiro

    2006-08-01

    Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as

  5. Sox17-Mediated XEN Cell Conversion Identifies Dynamic Networks Controlling Cell-Fate Decisions in Embryo-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Angela C.H. McDonald

    2014-10-01

    Full Text Available Little is known about the gene regulatory networks (GRNs distinguishing extraembryonic endoderm (ExEn stem (XEN cells from those that maintain the extensively characterized embryonic stem cell (ESC. An intriguing network candidate is Sox17, an essential transcription factor for XEN derivation and self-renewal. Here, we show that forced Sox17 expression drives ESCs toward ExEn, generating XEN cells that contribute to ExEn when placed back into early mouse embryos. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. To orchestrate this conversion process, Sox17 acts in autoregulatory and feedforward network motifs, regulating dynamic GRNs directing cell fate. Sox17-mediated XEN conversion helps to explain the regulation of cell-fate changes and reveals GRNs regulating lineage decisions in the mouse embryo.

  6. Interphase death of dividing cells. Death rate of cultured Chinese hamster fibroblasts as a function of ph inside and outside cells

    International Nuclear Information System (INIS)

    Veksler, A.M.; Kublik, L.N.; Ehjdus, L.Kh.

    1990-01-01

    In studying interphase death (ID) of dividing cells from Chinese hamster fibroblast culture a differently directed relationship between ID rate and pH has been shown: the ID rate increases with pH increasing from 6.6 to 8.1 and decreases with pH from 5.0 to 6.6. The dependence is the same as that observed with lymphoid cells. With radiation doses increasing from 100 to 600 Gy and pH defined, the ID rate increases

  7. Modification of the repair of potentially lethal damage in plateau-phase Chinese hamster cells by 2-chlorodeoxyadenosine

    Energy Technology Data Exchange (ETDEWEB)

    Tanabe, Kiyoshi; Hiraoka, Wakako; Kuwabara, Mikinori; Matsuda, Akira; Ueda, Tohru; Sato, Fumiaki.

    1988-09-01

    The ability of 2-chlorodeoxyadenosine, a ribonucleotide reductase inhibitor, to inhibit the repair of potentially lethal damage was demonstrated in Chinese hamster V79 cells after X irradiation in plateau-phase cultures. This ability of the drug was completely diminished when deoxycytidine was added at the same time, though this was slightly affected by the addition of adenosine, suggesting that this drug was phosphorylated by deoxycytidine kinase to serve as an inhibitor of the repair of potentially lethal damage. Compared with hydroxyurea, another ribonucleotide reductase inhibitor, this drug appeared to contain its own activity which suppressed the repair of potentially lethal damage. A combined study of post-irradiation treatment with hypertonic salt solution and with this drug on the fixation of potentially lethal damage revealed that this drug inhibited the repair of hypertonic-insensitive potentially lethal damage.

  8. Modification of the repair of potentially lethal damage in plateau-phase Chinese hamster cells by 2-chlorodeoxyadenosine

    International Nuclear Information System (INIS)

    Tanabe, Kiyoshi; Hiraoka, Wakako; Kuwabara, Mikinori; Matsuda, Akira; Ueda, Tohru; Sato, Fumiaki.

    1988-01-01

    The ability of 2-chlorodeoxyadenosine, a ribonucleotide reductase inhibitor, to inhibit the repair of potentially lethal damage was demonstrated in Chinese hamster V79 cells after X irradiation in plateau-phase cultures. This ability of the drug was completely diminished when deoxycytidine was added at the same time, though this was slightly affected by the addition of adenosine, suggesting that this drug was phosphorylated by deoxycytidine kinase to serve as an inhibitor of the repair of potentially lethal damage. Compared with hydroxyurea, another ribonucleotide reductase inhibitor, this drug appeared to contain its own activity which suppressed the repair of potentially lethal damage. A combined study of post-irradiation treatment with hypertonic salt solution and with this drug on the fixation of potentially lethal damage revealed that this drug inhibited the repair of hypertonic-insensitive potentially lethal damage. (author)

  9. Mutagenic and epigenetic influence of caffeine on the frequencies of UV-induced ouabain-resistant Chinese hamster cells

    International Nuclear Information System (INIS)

    Chang, Chia-Cheng; Philipps, C.; Trosko, J.E.; Hart, R.W.

    1977-01-01

    Caffeine, given as a post-treatment to UV-irradiated Chinese hamster cells in vitro, modified the frequency of induced mutations at the ouabain resistance locus. Mutation frequencies were increased when caffeine was added only for the DNA repair and mutation fixation period. When caffeine was added after the DNA repair and mutation fixation period, or immediately after DNA damage and for the entire repair and selection period, mutation frequencies were reduced. A hypothesis, given to explain both results, is that caffeine, by blocking a constitutive 'error-free' postreplication repair process, allows an 'error-prone' DNA repair process to produce many mutations. Moreover, caffeine, possibly by modifying C-AMP metabolism, causes a repression of induced mutations which, in effect, explains its anti-mutagenic and anti-carcinogenic properties

  10. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  11. Hyperthermic survival of Chinese hamster ovary cells as a function of cellular population density at the time of plating

    International Nuclear Information System (INIS)

    Highfield, D.P.; Holahan, E.V.; Holahan, P.K.; Dewey, W.C.

    1984-01-01

    The survival of synchronous G 1 or asynchronous Chinese hamster ovary cells in vitro to heat treatment may depend on the cellular population density at the time of heating and/or as the cells are cultured after heating. The addition of lethally irradiated feeder cells may increase survival at 10 -3 by as much as 10- to 100-fold for a variety of conditions when cells are heated either in suspension culture or as monolayers with or without trypsinization. The protective effect associated with feeder cells appears to be associated with close cell-to-cell proximity. However, when cells are heated without trypsinization about 24 hr or later after plating, when adaptation to monolayer has occurred, the protective effect is reduced; i.e., addition of feeder cells enhances survival much less, for example, about 2- to 3-fold at 10 -2 -10 -3 survival. Also, the survival of a cell to heat is independent of whether the neighboring cell in a microcolony is destined to live or die. Finally, if protective effects associated with cell density do occur and are not controlled, serious artifacts can result as the interaction of heat and radiation is studied; for example, survival curves can be moved upward, and thus changed in shape as the number of cells plated is increased with an increase in the hyperthermic treatment or radiation dose following hyperthermia. Therefore, to understand mechanisms and to obtain information relevant to populations of cells in close proximity, such as those in vivo, these cellular population density effects should be considered and understood

  12. Inmunogenicidad y capacidad protectora en hamsters de vacunas antileptospirósicas monovalentes de células enteras del serogrupo Ballum Immunogenicity and protective capacity of leptospiral whole-cell monovalent serogroup Ballum vaccines in hamsters

    Directory of Open Access Journals (Sweden)

    A. González

    2005-12-01

    Full Text Available El serogrupo Ballum de Leptospira constituye en la actualidad la primera causa de leptospirosis humana en Cuba. Vacunas de células enteras químicamente inactivadas fueron formuladas a partir de dos cepas clínicas de Leptospira interrogans serogrupo Ballum empleando como adyuvante hidróxido de aluminio. Los niveles de aglutininas inducidos en hamsters por una u otra preparación vacunal fueron estimados mediante aglutinación microscópica y la actividad IgG específica fue cuantificada mediante ELISA. La capacidad de protección homóloga y heteróloga contra la infección letal y subletal se determinó mediante el desafío con 100 y 10 000 DL50 de cinco cepas virulentas pertenecientes a los serogrupos Ballum, Canicola, Icterohaemorrhagiae y Pomona. Las evaluaciones realizadas demostraron que ambas vacunas fueron inmunogénicas e indujeron una completa protección homóloga en el modelo animal empleado. La protección cruzada frente a serogrupos heterólogos solo fue significativa en una de las preparaciones monovalentes frente al desafío con 100 DL50 de Canicola. Como resultado de este estudio se pudo comprobar la alta inmunogenicidad y capacidad protectora en hamsters de vacunas monovalentes de células enteras formuladas a partir de dos cepas candidatas vacunales del serogrupo de Leptospira de mayor circulación en humanos en Cuba no incluido en la vacuna actualmente disponible.Leptospira serogroup Ballum is at present the first cause of human leptospirosis in Cuba. Killed whole-cell vaccines were formulated with two clinical isolates of Leptospira interrogans serogroup Ballum using aluminum hydroxide as adjuvant. Agglutinins levels induced by each vaccine in hamsters were estimated by microscopic agglutination test and specific IgG activities were quantified by a whole cell-based enzyme-linked immunosorbent assay. Homologous and cross protective capacity against lethal and sublethal infection were determined in vaccinated animals by

  13. No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

    Science.gov (United States)

    Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

    2010-01-01

    Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

  14. Effect of ambient temperature on the proliferation of brown adipocyte progenitors and endothelial cells during postnatal BAT development in Syrian hamsters.

    Science.gov (United States)

    Nagaya, Kazuki; Okamatsu-Ogura, Yuko; Nio-Kobayashi, Junko; Nakagiri, Shohei; Tsubota, Ayumi; Kimura, Kazuhiro

    2018-04-02

    In Syrian hamsters, brown adipose tissue (BAT) develops postnatally through the proliferation and differentiation of brown adipocyte progenitors. In the study reported here, we investigated how ambient temperature influenced BAT formation in neonatal hamsters. In both hamsters raised at 23 or 30 °C, the interscapular fat changed from white to brown coloration in an age-dependent manner and acquired the typical morphological features of BAT by day 16. However, the expression of uncoupling protein 1, a brown adipocyte marker, and of vascular endothelial growth factor α were lower in the group raised at 30 °C than in that raised at 23 °C. Immunofluorescent staining revealed that the proportion of Ki67-expressing progenitors and endothelial cells was lower in the 30 °C group than in the 23 °C group. These results indicate that warm ambient temperature suppresses the proliferation of brown adipocyte progenitors and endothelial cells and negatively affects the postnatal development of BAT in Syrian hamsters.

  15. In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

    Science.gov (United States)

    Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.

  16. Isolation and characterization of a Chinese hamster ovary cell line deficient in fatty alcohol:NAD+ oxidoreductase activity

    International Nuclear Information System (INIS)

    James, P.F.; Lee, J.; Rizzo, W.B.; Zoeller, R.A.

    1990-01-01

    The authors have isolated a mutant Chinese hamster ovary cell line that is defective in long-chain fatty alcohol oxidation. The ability of the mutant cells to convert labeled hexadecanol to the corresponding fatty acid in vivo was reduced to 5% of the parent strain. Whole-cell homogenates from the mutant strain, FAA.1, were deficient in long-chain fatty alcohol:NAD + oxidoreductase activity, which catalyzes the oxidation of hexadecanol to hexadecanoic acid, although the intermediate fatty aldehyde was formed normally. A direct measurement of fatty aldehyde dehydrogenase showed that the FAA.1, strain was defective in this component of FAO activity. FAA.1 is a two-stage mutant that was selected from a previously described parent strain, ZR-82, which is defective in ether lipid biosynthesis and peroxisome assembly. Because of combined defects in ether lipid biosynthesis and fatty alcohol oxidation, the ability of the FAA.1 cells to incorporate hexadecanol into complex lipids was greatly impaired, resulting in a 60-fold increase in cellular fatty alcohol levels. As the FAO deficiency in FAA.1 cells appears to be identical to the defect associated with the human genetic disorder Sjoegren-Larsson syndrome, the FAA.1 cell line may be useful in studying this disease

  17. Mutation to ouabain-resistance in Chinese hamster cells: induction by ethyl methanesulphonate and lack of induction by ionising radiation

    International Nuclear Information System (INIS)

    Thacker, J.; Stephens, M.A.; Stretch, A.

    1978-01-01

    The spontaneous frequency of mutants resistant to growth inhibition by ouabian (OUAsup(R) mutants) was found to be about 5.10 -5 per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1.10 -6 in 1 mM ouabain. No increase in the frequency of OUAsup(R) mutants was found in cultures treated with γ-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than γ-rays for the induction of thioguanine-resistant mutants in these cells. Recent evidence is reviewed in support of the suggestion that ionising radiation is unable to induce OUAsup(R) mutants because of the severity of the genetic damage it causes. (Auth.)

  18. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. III. Genetic evidence for utilization of phosphatidylcholine and phosphatidylethanolamine as precursors

    International Nuclear Information System (INIS)

    Kuge, O.; Nishijima, M.; Akamatsu, Y.

    1986-01-01

    We reported that Chinese hamster ovary (CHO) cells contain two different serine-exchange enzymes (I and II) which catalyze the base-exchange reaction of phospholipid(s) with serine and that a phosphatidylserine-requiring mutant (strain PSA-3) of CHO cells is defective in serine-exchange enzyme I and lacks the ability to synthesize phosphatidylserine. In this study, we examined precursor phospholipids for phosphatidylserine biosynthesis in CHO cells. When mutant PSA-3 and parent (CHO-K1) cells were cultured with [ 32 P]phosphatidylcholine, phosphatidylserine in the parent accumulated radioactivity while that in the mutant was not labeled significantly. On the contrary, when cultured with [ 32 P]phosphatidylethanolamine, the mutant incorporated the label into phosphatidylserine more efficiently than the parent. Furthermore, we found that mutant PSA-3 grew normally in growth medium supplemented with 30 microM phosphatidylethanolamine as well as phosphatidylserine and that the biosynthesis of phosphatidylserine in the mutant was normal when cells were cultured in the presence of exogenous phosphatidylethanolamine. The simplest interpretation of these findings is that phosphatidylserine in CHO cells is biosynthesized through the following sequential reactions: phosphatidylcholine----phosphatidylserine----phosphatidylethanolamine--- - phosphatidylserine. The three reactions are catalyzed by serine-exchange enzyme I, phosphatidylserine decarboxylase, and serine-exchange enzyme II, respectively

  19. Effects of turmeric and its active principle, curcumin, on bleomycin-induced chromosome aberrations in Chinese hamster ovary cells

    Directory of Open Access Journals (Sweden)

    Araújo Maria Cristina P.

    1999-01-01

    Full Text Available Naturally occurring antioxidants have been extensively studied for their capacity to protect organisms and cells from oxidative damage. Many plant constituents including turmeric and curcumin appear to be potent antimutagens and antioxidants. The effects of turmeric and curcumin on chromosomal aberration frequencies induced by the radiomimetic agent bleomycin (BLM were investigated in Chinese hamster ovary (CHO cells. Three concentrations of each drug, turmeric (100, 250 and 500 mg/ml and curcumin (2.5, 5 and 10 mg/ml, were combined with BLM (10 mg/ml in CHO cells treated during the G1/S, S or G2/S phases of the cell cycle. Neither turmeric nor curcumin prevented BLM-induced chromosomal damage in any phases of the cell cycle. Conversely, a potentiation of the clastogenicity of BLM by curcumin was clearly observed in cells treated during the S and G2/S phases. Curcumin was also clastogenic by itself at 10 µg/ml in two protocols used. However, the exact mechanism by which curcumin produced clastogenic and potentiating effects remains unknown.

  20. Persistence of sister chromatid exchanges and in vitro morphological transformation of Syrian hamster fetal cells by chemical and physical carcinogens

    International Nuclear Information System (INIS)

    Popescu, N.C.; Amsbaugh, S.C.; DiPaolo, J.A.

    1985-01-01

    The induction of neoplastic cell transformation is closely associated with DNA alterations which occur shortly after carcinogen exposure. Sister chromatid exchange (SCE) formation is a sensitive indicator of carcinogen-DNA interaction and correlates with the induction of morphological cell transformation. The persistence of lesions generating SCE produced by chemical and physical carcinogens and its relevance to the induction of morphologic transformation was evaluated in coordinated experiments with cultured Syrian hamster fetal cells (HFC). Exponentially growing HFC were exposed for 1 h to benzo[a]pyrene (BP), methyl-methanesulfonate (MMS), cis-platinum (II) diaminedichloride (cis Pt II), N-methyl-N'-nitrosourea (MNU), mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-acetoxy-2-fluorenyl-acetamide (AcAAF) or u.v. light irradiated. SCE analysis demonstrates that for a period of 48 h after carcinogen exposure, during which time the cells undergo at least four replicative cycles, DNA damage generating SCE induced by all chemical carcinogens either persisted or was partially removed, whereas u.v.-induced lesions were completely removed. An elevated SCE frequency persisted after two additional cell cycles after treatment with BP, AcAAF or MMC without increased cell lethality as compared to other carcinogens whose lesions were completely eliminated during the same period

  1. Early bovine embryos regulate oviduct epithelial cell gene expression during in vitro co-culture.

    Science.gov (United States)

    Schmaltz-Panneau, Barbara; Cordova, Amanda; Dhorne-Pollet, Sophie; Hennequet-Antier, Christelle; Uzbekova, Sveltlana; Martinot, Emmanuelle; Doret, Sarah; Martin, Patrice; Mermillod, Pascal; Locatelli, Yann

    2014-10-01

    In mammals, the oviduct may participate to the regulation of early embryo development. In vitro co-culture of early bovine embryos with bovine oviduct epithelial cells (BOEC) has been largely used to mimic the maternal environment. However, the mechanisms of BOEC action have not been clearly elucidated yet. The aim of this study was to determine the response of BOEC cultures to the presence of developing bovine embryos. A 21,581-element bovine oligonucleotide array was used compare the gene expression profiles of confluent BOEC cultured for 8 days with or without embryos. This study revealed 34 differentially expressed genes (DEG). Of these 34 genes, IFI6, ISG15, MX1, IFI27, IFI44, RSAD2, IFITM1, EPSTI1, USP18, IFIT5, and STAT1 expression increased to the greatest extent due to the presence of embryos with a major impact on antiviral and immune response. Among the mRNAs at least 25 are already described as induced by interferons. In addition, transcript levels of new candidate genes involved in the regulation of transcription, modulation of the maternal immune system and endometrial remodeling were found to be increased. We selected 7 genes and confirmed their differential expression by quantitative RT-PCR. The immunofluorescence imaging of cellular localization of STAT1 protein in BOEC showed a nuclear translocation in the presence of embryos, suggesting the activation of interferon signaling pathway. This first systematic study of BOEC transcriptome changes in response to the presence of embryos in cattle provides some evidences that these cells are able to adapt their transcriptomic profile in response to embryo signaling. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Activation of ribosomal RNA genes in porcine embryos produced in vitro or by somatic cell nuclear transfer

    DEFF Research Database (Denmark)

    Bjerregaard, Bolette; Pedersen, Hanne Gervi; Jakobsen, Anne Sørig

    2007-01-01

    The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell...

  3. Cell-type specific increases in female hamster nucleus accumbens spine density following female sexual experience.

    Science.gov (United States)

    Staffend, Nancy A; Hedges, Valerie L; Chemel, Benjamin R; Watts, Val J; Meisel, Robert L

    2014-11-01

    Female sexual behavior is an established model of a naturally motivated behavior which is regulated by activity within the mesolimbic dopamine system. Repeated activation of the mesolimbic circuit by female sexual behavior elevates dopamine release and produces persistent postsynaptic alterations to dopamine D1 receptor signaling within the nucleus accumbens. Here we demonstrate that sexual experience in female Syrian hamsters significantly increases spine density and alters morphology selectively in D1 receptor-expressing medium spiny neurons within the nucleus accumbens core, with no corresponding change in dopamine receptor binding or protein expression. Our findings demonstrate that previous life experience with a naturally motivated behavior has the capacity to induce persistent structural alterations to the mesolimbic circuit that can increase reproductive success and are analogous to the persistent structural changes following repeated exposure to many drugs of abuse.

  4. Effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development

    Directory of Open Access Journals (Sweden)

    Coyral-Castel Stéphanie

    2010-03-01

    Full Text Available Abstract Background Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor in bovine ovary and its role on ovarian cells and embryo, remain however to be determined. Methods Here, we identified the adiponectin system in bovine ovarian cells and embryo using RT-PCR, immunoblotting and immunohistochemistry. Furthermore, we investigated in vitro the effects of recombinant human adiponectin (10 micro g/mL on proliferation of granulosa cells (GC measured by [3H] thymidine incorporation, progesterone and estradiol secretions measured by radioimmunoassay in the culture medium of GC, nuclear oocyte maturation and early embryo development. Results We show that the mRNAs and proteins for the adiponectin system are present in bovine ovary (small and large follicles and corpus luteum and embryo. Adiponectin, AdipoR1 and AdipoR2 were more precisely localized in oocyte, GC and theca cells. Adiponectin increased IGF-1 10(-8 M-induced GC proliferation (P Conclusions In bovine species, adiponectin decreased insulin-induced steroidogenesis and increased IGF-1-induced proliferation of cultured GC through a potential involvement of ERK1/2 MAPK pathway, whereas it did not modify oocyte maturation and embryo development in vitro.

  5. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Jin Li

    Full Text Available Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf. Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.

  6. Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos

    DEFF Research Database (Denmark)

    Jakobsen, Anne Sørig; Avery, Birthe; Dieleman, Steph J.

    2006-01-01

    Transcription from the embryos own ribosomal genes is initiated in most species at the same time as the maternal-embryonic transition. Recently data have indicated that a minor activation may take place during the third embryonic cell cycle in the bovine, one cell cycle before the major activation...

  7. Inhibition of replicon initiation and DNA elongation in Chinese hamster ovary cells by treatment at 45.5 degrees C

    International Nuclear Information System (INIS)

    Wong, R.S.; Dewey, W.C.

    1982-01-01

    Heat treatment of Chinese hamster ovary cells at 45.5 degrees C for 15 minutes resulted in the inhibition of both the replicon initiation and the DNA elongation processes. Analysis of the DNA made after treatment showed that for up to 30 minutes after hyperthermia, there was a significant increase (45-80% above control level) in the amount of labeled DNA less than or equal to 40S in size and having a distinct peak of 20S. Therefore, elongation of 20S molecules into larger molecules was inhibited or slowed down. These small molecules did not accumulate when recovery times were longer than 30 minutes. The DNA made after 120 and 240 minutes postheat incubation was larger than control size and indicated that, although replicon initiation was still inhibited, elongation between replicons into 120S molecules could take place. However, their subsequent elongation into parental-size molecules was inhibited. The same delay in DNA elongation seen in cells examined immediately after treatment was still observed in cells heated and allowed to recover for 30 minutes. Also, after 30 minutes of recovery, heated cells still had more newly synthesized DNA in the single-stranded fraction than did control cells, which indicates that DNA elongation within a replicon is delayed for at least 30 minutes after heating. Furthermore, at 4 hours after heating, the inhibition of elongation of clusters of replicons into parental molecules prevailed

  8. Effects of ultraviolet irradiation on the rate and sequence of DNA replication in synchronized Chinese hamster cells

    International Nuclear Information System (INIS)

    Meyn, R.E.; Hewitt, R.R.; Thomson, L.F.; Humphrey, R.M.

    1976-01-01

    The effects of ultraviolet light (uv) irradiation on the rate of DNA replication in synchronized Chinese hamster ovary (CHO) cells were investigated. A technique for measuring semiconservative DNA replication was employed that involved growing the cells in medium containing 5-bromodeoxyuridine and subsequently determining the amount of DNA that acquired hybrid buoyant density in CsCl density gradients. One of the advantages of this technique was that it allowed a characterization of the extent of DNA replication as well as rate after irradiation. It was found that while there was a dose-dependent reduction in the rate of DNA replication following uv-irradiation, doses of up to 10 J/m 2 (which produce many dimers per replicon) did not prevent the ultimate replication of the entire genome. Hence, we conclude that dimers cannot be absolute blocks to DNA replication. In order to account for the total genome replication observed, a mechanism must exist that allows genome replication between dimers. The degree of reduction in the rate of replication by uv was the same whether the cells were irradiated at the Gl-S boundary or 1 h into S-phase. Previous work had shown that cells in early S-phase are considerably more sensitive to uv than cells at the G1-S boundary. Experiments specifically designed to test for reiterative replication showed that uv does not induce a second round of DNA replication within the same S-phase

  9. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    Energy Technology Data Exchange (ETDEWEB)

    Fendrick, J.L.; Iglewski, W.J. (Univ. of Rochester, NY (USA))

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.

  10. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    International Nuclear Information System (INIS)

    Fendrick, J.L.; Iglewski, W.J.

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [ 32 P]orthophosphate and 105 (vol/vol) fetal bovine serum. A 32 P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32 P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32 P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [ 32 P]AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32 P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and [ 32 P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32 P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32 P label was lost from the EF-2 within 30 min

  11. Permeability changes and incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of Chinese hamsters affected by MEA and low temperature

    International Nuclear Information System (INIS)

    Ermekova, V.M.; Kondakova, N.V.; Levitman, M.Kh.; Saugabaeva, K.M.; Ehjdus, L.Kh.

    1976-01-01

    Action of MEA and low temperature (20degC) on the incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of chinese hamsters has been studied. It has been found that each of the above-mentioned factors equally decreases the label uptake into the cell and DNA. It is concluded that MEA and low temperature do not substantially influence the rate of DNA synthesis

  12. The influence of continuous γ-irradiation at decreasing dose-rate on the survival rote and induction of gene mutations in cultured Chinese hamster cells

    International Nuclear Information System (INIS)

    Feoktistova, T.P.; Elisova, E.V.; Stavrakova, N.M.

    1991-01-01

    Continuous γ-irradiation at decreasing dose-rate was shown to be less effective than acute exposure with regard to the lethal effect and frequency of mutations of resistance to 6-thioguanine in cultured Chinese hamster cells. The cell population subjected to continuons irradiation was d more radioresistant than the intact one. Lethal and genetic effects of continuous irradiation at decreasing dose-rate were mainly determined by the contribution of the radiation dose received during the first 24 h of exposure

  13. Characterization of membrane lipid fluidity in human embryo cells malignantly transfer med post 238Pu α irradiation

    International Nuclear Information System (INIS)

    Qi Zirong; Sun Ling; Liu Guolian; Shen Zhiyuan

    1992-01-01

    The membrane lipid fluidity of malignantly transformed human embryo cells following 238 Pu α particlce irradiation in vitro has been studied. The results indicate that the ontogenesis depends on irradiation dose (Gy) and the membrane lipid fluidity in malignantly transformed cells is higher than that in normal embryo cells. With the microviscosity (η) of cells plotted against the cell counts, the correlation coefficient (γ) is calculated to be between 0.9936 and 0.9999. Since the malignant transformation of irradiated embryo cells is manifested early on cell membrane lipid, the fluidity of membrane lipid can be used as an oncologic marker

  14. Isolation and culture of porcine neural progenitor cells from embryos and pluripotent stem cells

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Hall, Vanessa Jane; Hyttel, Poul

    2013-01-01

    from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month......The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement...... therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs...

  15. Control of protein synthesis in cell-free extracts of sea urchin embryos

    International Nuclear Information System (INIS)

    Hansen, L.J.; Huang, W.I.; Jagus, R.

    1986-01-01

    Although the increase in protein synthesis that occurs after fertilization of sea urchin eggs results from increased utilization of stored maternal mRNA, the underlying mechanism is unknown. The authors have prepared cell-free extracts from S.purpuratus and A.puctulata unfertilized eggs and 2-cell embryos that retain the protein synthetic differences observed in vivo. The method is based on that of Dr. Alina Lopo. 35 S methionine incorporation is linear during a 30 min incubation and is 10-20 fold higher in extracts from 2-cell embryos than unfertilized eggs. Addition of purified mRNA does not stimulate these systems, suggesting a regulatory mechanism other than mRNA masking. Addition of rabbit reticulocyte ribosomal salt wash stimulated protein synthesis in extracts from eggs but not embryos, suggesting deficiencies in translational components in unfertilized eggs. Mixing of egg and embryo lysates indicated the presence of a weak protein synthesis inhibitor in eggs. Translational control in developing sea urchin embryos thus appears to be complex, involving both stimulatory and inhibitory factors

  16. Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

    Directory of Open Access Journals (Sweden)

    Tony Y-C Tsai

    2014-02-01

    Full Text Available During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min and the subsequent 11 cycles are short (∼30 min and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

  17. Caspase activity and expression of cell death genes during development of human preimplantation embryos.

    Science.gov (United States)

    Spanos, S; Rice, S; Karagiannis, P; Taylor, D; Becker, D L; Winston, R M L; Hardy, K

    2002-09-01

    It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.

  18. The diversity of nanos expression in echinoderm embryos supports different mechanisms in germ cell specification.

    Science.gov (United States)

    Fresques, Tara; Swartz, Steven Zachary; Juliano, Celina; Morino, Yoshiaki; Kikuchi, Mani; Akasaka, Koji; Wada, Hiroshi; Yajima, Mamiko; Wessel, Gary M

    2016-07-01

    Specification of the germ cell lineage is required for sexual reproduction in all animals. However, the timing and mechanisms of germ cell specification is remarkably diverse in animal development. Echinoderms, such as sea urchins and sea stars, are excellent model systems to study the molecular and cellular mechanisms that contribute to germ cell specification. In several echinoderm embryos tested, the germ cell factor Vasa accumulates broadly during early development and is restricted after gastrulation to cells that contribute to the germ cell lineage. In the sea urchin, however, the germ cell factor Vasa is restricted to a specific lineage by the 32-cell stage. We therefore hypothesized that the germ cell specification program in the sea urchin/Euechinoid lineage has evolved to an earlier developmental time point. To test this hypothesis we determined the expression pattern of a second germ cell factor, Nanos, in four out of five extant echinoderm clades. Here we find that Nanos mRNA does not accumulate until the blastula stage or later during the development of all other echinoderm embryos except those that belong to the Echinoid lineage. Instead, Nanos is expressed in a restricted domain at the 32-128 cell stage in Echinoid embryos. Our results support the model that the germ cell specification program underwent a heterochronic shift in the Echinoid lineage. A comparison of Echinoid and non-Echinoid germ cell specification mechanisms will contribute to our understanding of how these mechanisms have changed during animal evolution. © 2016 Wiley Periodicals, Inc.

  19. Chromosomal aberrations of the Chinese hamster cell line V79 after irradiation with X-rays and heavy ions

    International Nuclear Information System (INIS)

    Mueller, W.

    1985-02-01

    The study on hand examines chromosomal aberrations in Chinese hamster 79 cells. Irradiation involved a number of heavy ions ranging from neon to uranium with an energy variation between 0.3 and 20 MeV/u. Linear energy transfer ranged from 270 to 16,300 keV/μm. X-ray tests were run for reasons of comparison. Experiments showed the following results: 1) Aberration rate increases in dependence of nuclear charge number or LET resp. 2) The distribution of the chromosome-damage instances found differed markedly from corresponding measurements following irradiation with thinly ionizing radiation. In contrast to x-irradiation, it is possible, therefore, to obtain high aberration yields in preparations made immediately after irradiation. 3) The maximum of aberration yield after heavy-ion irradiation could be shown to occur as early as 4h after irradiation. This is true in x-irradiation for but small doses. 4) The radiation-sensitizing effect of caffeine and its action on the repair system of the cell could be confirmed for x-irradiation and could be described for heavy ions for the first time. 5) The radiation-protection effect of cysteamine could be re-affirmed for thinly ionizing radiation, however, it could not be verified for heavy ions. 6) Irradiation of cells by means of particles of a defined range supports the hypothesis that the particularly radiation-sensitive regions of the nucleus membrane constitute the cell's crucial target. (orig./MG) [de

  20. DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Romac, S; Leong, P; Sockett, H; Hutchinson, F [Yale Univ., New Haven, CT (USA). Dept. of Molecular Biophysics and Biochemistry

    1989-09-20

    The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Excherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt{sup -} colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR) and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes. (author).

  1. Effects of hyperthermia and x irradiation on sister chromatid exchange (SCE) frequency in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Livingston, G.K.; Dethlefsen, L.A.

    1979-01-01

    The BrdUrd labeling method was used to evaluate the effects of hyperthermia, x irradiation, and the combined treatment on the incidence of sister chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells. Cells cultured in McCoy's 5A media containing 10 μM 5-bromodeoxyuridine were synchronized after one cell cycle by mitotic shake-off. Early-G 1 cells were heated by submerging culture flasks in a 44 +- 0.05 0 C water bath for periods of 20, 40, and 60 min. By the same method, other cultures were x irradiated at doses of 100, 200, 400, and 600 rad. A third protocol involved combined treatment of 20 min at 44 0 C followed immediately by one of the above radiation doses. A fourth protocol reversed the sequence of the combined treatment applying x irradiation (200 or 400 rad) followed immediately by hyperthermia. The data showed that hyperthermia and x irradiation both elevated the frequency of SCEs significantly whether applied separately or together. The combined treatment (heat: 20 min at 44 0 C plus varying x-radiation doses) produced results suggestive of a synergistic interaction. The sequence of the heat and x irradiation did not appear to have a significant effect on the production of SCE

  2. Similar kinetics of chromatid aberrations in X-irradiated xrs 5 and wild-type Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    MacLeod, R.A.F.; Bryant, P.E.

    1990-01-01

    We have studied the kinetics of chromatid aberrations in cells of the Chinese hamster ovary (CHO-K1) derived, X-ray sensitive cell line xrs 5 irradiated in the G 2 phase at 37 0 C, as well as during a cell cycle extended by transient hypothermia at 33 0 C. While a given X-ray dose was estimated to produce about 4 times as many chromatid break and twice the frequency of exchanges in xrs 5 cells as in the parent line, there was no difference between the lines in the rates of disappearance of chromatid breaks during G 2 at either temperature; and similar patterns of chromatid exchange kinetics were observed in the two lines. Both the frequencies and distributions of chromatid breaks at different times after irradiation are consistent with the view that the disappearance of these during incubation represents a repair process. These results imply that the G 2 chromosomal radiosensitivity of the xrs 5 mutant resides at the level of initial chromatid damage. (author)

  3. Influences of somatic donor cell sex on and embryo development following somatic cell nuclear transfer in pigs

    Directory of Open Access Journals (Sweden)

    Jae-Gyu Yoo

    2017-04-01

    Full Text Available Objective The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8 was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT groups (31.4±8.3 to 33.4±11.1. After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05 between sexes. Conclusion The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

  4. The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

    DEFF Research Database (Denmark)

    Morovic, Martin; Murin, Matej; Strejcek, Frantisek

    2016-01-01

    in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early......One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription....... In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly...

  5. Contribution of Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells to Chimeras through Injection and Coculture of Embryos

    OpenAIRE

    Guo, Jitong; Wu, Baojiang; Li, Shuyu; Bao, Siqin; Zhao, Lixia; Hu, Shuxiang; Sun, Wei; Su, Jie; Dai, Yanfeng; Li, Xihe

    2014-01-01

    Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cel...

  6. Induction of DNA-protein cross-linking in Chinese hamster cells by monochromatic 365 and 405 NM ultraviolet light

    International Nuclear Information System (INIS)

    Han, A.; Peak, M.J.; Peak, J.G.

    1984-01-01

    The survival, the induction of DNA-protein cross-linking, and the number of T4-endonuclease sensitive sites were measured in Chinese hamster cells that had been irradiated with 365 and 405 nm monochromatic light. The survival measurements show that cells are somewhat less sensitive to 405 nm light than to 365 nm light. The difference is expressed predominantly in the shoulder widths of the survival curves, whereas the slopes of the two curves are about the same. Induction of pyrimidine dimers, as indicated by the number of endonuclease-sensitive sites, after exposures that produce about 10% survival is very low at 365 nm (approx. 4 endonuclease sites per 2 x 10 8 daltons), while no dimers are detected at 405 nm. In contrast, DNA-protein cross-links are induced rather effectively at either wavelength even after exposures that result in a relatively high survival (60-20%). These measurements support the conclusion that lethality in mammalian cells after irradiations with 365 or 405 nm light is caused by a nondimer damage, possibly DNA-protein cross-links. (author)

  7. Modification of thermal sensitivity of Chinese hamster cells by exposure to solutions of monovalent and divalent cationic salts

    International Nuclear Information System (INIS)

    Raaphorst, G.P.; Azzam, E.I.; Vadasz, J.

    1984-06-01

    Chinese hamster V79 cells were heated in culture medium or in 0.155-mol.dm -3 solutions of LiCl, NaCl, KCl, MgCl 2 , CaCl 2 and BaCl 2 . The presence of any one of these ionic solutions during heating increased the thermal sensitivity of the cells. The order of increased thermal sensitivity was KCl > LiCl > NaCl for the monovalent salts and BaCl 2 > MgCl 2 > CaCl 2 for the divalent cation salts. The addition of glucose to LiCl or NaCl solutions did not reduce the thermal sensitization caused by these solutions. When cells were sensitized by LiCl or NaCl treatment, a change in pH from 7.2 to 6.6 did not further increase thermal sensitivity. These data show that nutrient and ionic factors and their interplay are involved in cellular thermal sensitivity

  8. Photodynamic inactivation of rubella virus enhances recombination with a latent virus of a baby hamster kidney cell line BHK21

    International Nuclear Information System (INIS)

    Yamamoto, Nobuto; Urade, Masahiro

    1989-01-01

    Rubella virus is very sensitive to photodynamic action. When tested with 1.2 x 10 -5 M toluidine blue and 8 W fluorescent lamp at a fluence of 11 W/m 2 , inactivation kinetics showed a linear single hit curve with a k value of 1.48 min -1 . Photodynamic inactivation of rubella virus greatly enhanced recombination with a latent virus (R-virus) of baby hamster kidney BHK21 cells. In contrast, no hybrids were detected in lysates of the cells infected with either UV-treated or untreated rubella virus. Therefore, hybrid viruses were readily detected only in lysates of BHK21 cells infected with photodynamically treated rubella virus. Photodynamic damage of rubella virus genomes generated a new hybrid type (hybrid type 3) in addition to a previously described type 2 hybrid (formerly designated as HPV-RV variant). Although both of these hybrid types carry the CF antigens of rubella virus, plaque forming ability of type 3 hybrid is neutralized neither by anti-rubella serum nor by anti-latent virus serum while type 2 hybrid is neutralized by anti-latent virus serum. (author)

  9. Poly(ADP-ribose) metabolism in X-irradiated Chinese hamster cells: its relation to repair of potentially lethal damage

    International Nuclear Information System (INIS)

    Ben-Hur, E.; Elkind, M.M.

    1984-01-01

    Nicotinamide-adenine dinucleotide (NAD + ) is the substrate used by cells in poly(ADP-ribose) synthesis. X-irradiation of log-phase Chinese hamster cells caused a rapid decrease in NAD + levels which was linearly dependent on radiation dose. The activity of ADP-ribosyl transferase (ADPRT) also increased linearly with radiation dose. The decrease of NAD + was slower, and the increase in ADPRT activity was less pronounced, in a radiation sensitive line, V79-AL162/S-10. An inhibitor of ADPRT, m-aminobenzamide, largely prevented the depletion of cellular NAD + and reduced the rate at which ADPRT activity disappeared during post-irradiation incubation. Post-irradiation treatment with hypertonic buffer or with medium containing D 2 O-which inhibit repair of radiation-induced potentially lethal damage-enhanced the depletion of NAD + and prevented the reduction in ADPRT activity following irradiation. The characteristics of the effects of treatment with hypertonic buffer on NAD + metabolism were qualitatively similar to the effects that such treatment has on radiation-induced cell killing. These results suggest that poly(ADP-ribose) synthesis after irradiation plays a role in the repair of potentially lethal damage. (author)

  10. Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

    International Nuclear Information System (INIS)

    Vikhanskaya, F.L.; Khrebtukova, I.A.; Manuilova, E.S.

    1985-01-01

    A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m 2 ) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m 2 ) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair. (orig.)

  11. Chromosome damage in Chinese hamster cells produced by 125I-UdR at the site of its incorporation

    International Nuclear Information System (INIS)

    Hughes, W.L.; Weinblatt, A.C.; Prensky, W.

    1978-01-01

    Metaphase chromosomal aberrations were produced by 125 I-labeled iododeoxyuridine ( 125 I-UdR) incorporated into Chinese hamster Don cells at the end of the S-period of the cell cycle. Chromosome damage and the number of autoradiographic silver grains were recorded for whole cells, for chromosome pairs 4 and 5 and for the X and the Y chromosomes. The X and the Y chromosomes, which label late in S, were at least twice as heavily labeled as chromosome pairs 4 and 5 - two readily recognizable autosomes of similar size. The incidence of chromosome damage was at least six times that which would have been expected from equivalent doses of X-rays and the incidence of damage was directly related to the number of silver grains over each chromosome. It is estimated that it takes four to ten disintegrations to produce a visible chromosome aberration. The finding that chromosome damage is localized at the site of the 125 I decay is most readily explained by the high flux of low energy Auger electrons occurring at the site of the decay of the incorporated 125 I atom. (Auth.)

  12. Response of the microtubular cytoskeleton following hyperthermia as a prognostic indicator of survival of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Coss, Ronald A.; Alden, Mark E.; Wachsberger, Phyllis R.; Smith, Nancy N.

    1996-01-01

    Purpose: The response of the microtubular (MT) cytoskeleton to hyperthermia was assessed as a prognostic indicator of cytotoxicity. Methods and Materials: Heat-induced collapse and subsequent recovery of the MT system were compared with survival for both nonthermotolerant (NT) and thermotolerant (TT) G1 populations of Chinese hamster ovary (CHO) cells. The response of the MT system was monitored using immunofluorescence staining. The G1 populations of NT and TT cells were heated by submersion in 45.0 and 43.0 deg. C waterbaths. Results: Heat-induced perinuclear collapse of the MT system did not correlate with survival for the NT and TT populations. However, recovery of the organization of the MT cytoskeleton was correlatable with survival. The regression line of survival plotted as a function of MT recovery is fit by: y = -0.43 + 1.03x, r 2 = 0.95 (p < 0.0005). Conclusion: Restoration of the organization of the MT cytoskeleton following hyperthermia may be used as a prognostic indicator of survival of CHO cells heated in G1

  13. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    International Nuclear Information System (INIS)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2014-01-01

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further

  14. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  15. Effects of D,L-buthionine-S,R-sulfoximine on cellular thiol levels and the oxygen effect in Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Astor, M.B.; Hall, E.J.; Biaglow, J.E.; Hartog, B.

    1984-01-01

    The role of glutathione (GSH) and total non-protein thiols (NPSH) in repairing radiation-induced free radical damage incurred under aerated and hypoxic conditions was investigated using Chinese hamster V79 cells cultured in vitro. GSH and NPSH levels were depleted in V79 cells of varying cell densities using the gamma-glutamyl-cysteine-synthetase inhibitor, D,L-Buthionine-S,R-sulfoximine (BSO). A small change in hypoxic cell radiosensitivity could be attributed to the loss of GSH while depletion of thiols to lower levels affected both aerated and hypoxic cell radiosensitivity, resulting in no change in the OER

  16. Effect of Wortmannin on the repair profiles of DNA double-strand breaks in the whole genome and in interstitial telomeric sequences of Chinese hamster cells

    International Nuclear Information System (INIS)

    Losada, Raquel; Rivero, Maria Teresa; Slijepcevic, Predrag; Goyanes, Vicente; Fernandez, Jose Luis

    2005-01-01

    The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was applied to analyze the effect of Wortmannin (WM) in the rejoining kinetics of ionizing radiation-induced DNA double-strand breaks (DSBs) in the whole genome and in the long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cell lines. The results indicate that the ITRS blocks from wild-type Chinese hamster cell lines, CHO9 and V79B, exhibit a slower initial rejoining rate of ionizing radiation-induced DSBs than the genome overall. Neither Rad51C nor the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) activities, involved in homologous recombination (HR) and in non-homologous end-joining (NHEJ) pathways of DSB repair respectively, influenced the rejoining kinetics within ITRS in contrast to DNA sequences in the whole genome. Nevertheless, DSB removal rate within ITRS was decreased in the absence of Ku86 activity, though at a lower affectation level than in the whole genome, thus homogenizing both rejoining kinetics rates. WM treatment slowed down the DSB rejoining kinetics rate in ITRS, this effect being more pronounced in the whole genome, resulting in a similar pattern to that of the Ku86 deficient cells. In fact, no WM effect was detected in the Ku86 deficient Chinese hamster cells, so probably WM does not add further impairment in DSB rejoining than that resulted as a consequence of absence of Ku activity. The same slowing effect was also observed after treatment of Rad51C and DNA-PKcs defective hamster cells by WM, suggesting that: (1) there is no potentiation of the HR when the NHEJ is impaired by WM, either in the whole genome or in the ITRS, and (2) that this impairment may probably involve more targets than DNA-PKcs. These results suggest that there is an intragenomic heterogeneity in DSB repair, as well as in the effect of WM on this process

  17. PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Jin, Long; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Zhang, Yu-Chen; Zhang, Guang-Lei; Xing, Xiao-Xu; Xuan, Mei-Fu; Luo, Qi-Rong; Yin, Xi-Jun; Kang, Jin-Dan

    2016-09-01

    To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12. Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed. PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

  18. Nucleoli from two-cell embryos support the development of enucleolated germinal vesicle oocytes in the pig.

    Science.gov (United States)

    Kyogoku, Hirohisa; Ogushi, Sugako; Miyano, Takashi

    2012-11-01

    Recent research has shown that nucleoli of oocytes at the germinal vesicle (GV) stage (GV nucleoli) are not necessary for oocyte maturation but are essential for early embryonic development. Nucleoli of 2-cell embryos (2-cell nucleoli) have morphology similar to that of nucleoli in oocytes at the GV stage. In this study, we examined the ability of 2-cell nucleoli to substitute for GV nucleoli in terms of supporting early embryonic development by nucleolus aspiration (enucleolation) and transfer into metaphase II (MII) oocytes or 2-cell embryos that were derived from enucleolated oocytes at the GV stage in the pig. When 2-cell embryos were centrifuged to move the lipid droplets to one side of the blastomere, multiple nucleoli in the nucleus fused into a single nucleolus. The nucleoli were then aspirated from the 2-cell embryos by micromanipulation. The injection of 2-cell nucleoli to GV enucleolated oocytes at the MII stage rescued the embryos from the early embryonic arrest, and the resulting oocytes developed to blastocysts. However, the injection of 2-cell and GV nucleoli to 2-cell embryos derived from GV enucleolated oocytes rarely restored the development to blastocysts. These results indicate that 2-cell nucleoli support early embryonic development as GV nucleoli and that the presence of nucleoli is essential for pig embryos before the 2-cell stage.

  19. Amount of sister chromatid exchanges and survival of Chinese hamster V79-4 cells after irradiation with 0,7 MeV neutrons

    International Nuclear Information System (INIS)

    Lapidus, I.L.; Nasonova, E.A.

    1987-01-01

    The dependence of the survival and induction of sister chromatid exchanges (SCEs) in Chinese hamster V79-4 cells on the dose of γ-rays and neutrons with average energy 0.7 MeV has been analysed. The value of RBE for neutrons was 5.5. It has been shown that the number of SCE increased with the dose of γ-irradiation and no induction could be detected after neutron irradiation

  20. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    International Nuclear Information System (INIS)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-01-01

    Highlights: → Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. → The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. → A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  1. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  2. Radioprotective action of WR-1065 on radiation-induced DNA strand breaks in cultured Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Murray, D.; VanAnkeren, S.C.; Milas, L.; Meyn, R.E.

    1988-01-01

    We have examined the radioprotective effect of WR-1065 on cultured Chinese hamster ovary cells. The effects of the drug on the induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) were measured using alkaline (pH 12.1) and neutral (pH 7.0) elution, respectively. Molecular protection factors (PFs) calculated from these data allowed us to determine whether the degree of modification of strand breakage accurately predicted the PFs measured using the biological end point of cell survival. The drug did protect against the induction of both SSBs and DSBs, although to an extent that did not appear to fully account for the degree of radioprotection in terms of cell killing measured under identical conditions. It is therefore unlikely that radioprotection by WR-1065 occurs simply as a consequence of a general lowering of all types of gamma-ray-induced DNA lesions, and it is possible that the drug could differentially protect against the induction of subsets of these DNA lesions. The rate of SSB rejoining was retarded following preirradiation treatment of cells with WR-1065, but there was no effect on DSB rejoining. Postirradiation treatment with WR-1065 also appeared to retard SSB rejoining but without an accompanying effect on either DSB rejoining or cell survival; however, this effect was largely reversed by the addition of catalase and was, therefore, probably a result of H 2 O 2 generated by autoxidation of the drug. Based on these observations, it would appear that the molecular actions of aminothiol radioprotective compounds that lead to reduced cell killing are much more complex than previously thought

  3. Autoradiographic study of protein synthesis recovery in root cells of Zea mays embryos during early stages of germination

    International Nuclear Information System (INIS)

    Deltour, Roger

    1977-01-01

    Recovery of protein synthesis was studied in primary root of germinating Zea mays embryos. [H 3 ] leucine or [H 3 ] lysine was provided for two hours at 16 0 C to embryos excised from kernels at various times after the beginning of germination. Protein synthesis (probably dependent on long-lived mRNA stocked in dormant embryo root cells) resumed during the first two hours of seed imbibition [fr

  4. Autoradiographic study of protein synthesis recovery in root cells of Zea mays embryos during early stages of germination

    Energy Technology Data Exchange (ETDEWEB)

    Deltour, R [Liege Univ. (Belgium)

    1977-05-02

    Recovery of protein synthesis was studied in primary root of germinating Zea mays embryos. (H/sup 3/) leucine or (H/sup 3/) lysine was provided for two hours at 16/sup 0/C to embryos excised from kernels at various times after the beginning of germination. Protein synthesis (probably dependent on long-lived mRNA stocked in dormant embryo root cells) resumed during the first two hours of seed imbibition.

  5. Laser-UV-microirradiation of Chinese hamster cells: the influence of the distribution of photolesions on unscheduled DNA synthesis

    International Nuclear Information System (INIS)

    Cremer, C.; Jabbur, G.

    1981-01-01

    Fibroblastoid Chinese hamster cells synchronized by mitotic selection were microirradiated in G1, using a low power laser-UV-microbeam (lambda = 257 nm). The incident energy was either concentrated on a small part of the nucleus (mode 1) or distributed over the whole nucleus (mode 11). Using the same incident UV energy, the local UV fluences were estimated to differ by two orders of magnitude. Following microirradiation the cells were incubated with [ 3 H]-thymidine for 2 h and thereafter processed for autoradiography. Silver grains were concentrated over the microirradiated part after mode 1 and distributed over the whole nucleus after mode 11 irradiation. To quantify the amount of unscheduled DNA synthesis, the number of grains per nucleus was determined. It increased with the total incident energy, but was not or only slightly affected by the mode of microirradiation, if appropriate autoradiographic conditions were used. The findings suggest that within the investigated range of energy densities (2.7-1000 J/m 2 ), the total amount of unscheduled DNA synthesis depends on the total number of pyrimidine dimers but not on their distribution in nuclear DNA. (author)

  6. Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation

    International Nuclear Information System (INIS)

    Ventura, A.M.; Meneghini, R.

    1984-01-01

    Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nonsubstituted DNA, was noticed in the density-distribution profiles. The density distribution of DNA along the gradient can provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement. (Auth.)

  7. Elastatinal and leupeptin: effects on u.v.-induced mutation and sister-chromatid exchanges in Chinese hamster cells

    International Nuclear Information System (INIS)

    Paul, P.; Fujiwara, Y.

    1981-01-01

    Microbial protease inhibitors elastatinal and leupeptin were tested for cytotoxicity and for effects on spontaneous and u.v.-induced 6-thioguanine-resistant (6TGsup(r)) mutation and sister-chromatid exchange (SCE) in V79 Chinese hamster cells. Continuous treatment with elastatinal exhibited marked cytotoxicity, while leupeptin was almost non-cytotoxic. Elastatinal rapidly induced cytotoxic effects as a function of its concentration and time of exposure. Near maximum cytotoxicity was reached after exposures of 6-8 h and this was partially abolished by the presence of 2.5 μg cycloheximide per ml. Concentrations of either protease inhibitor which gave 60-80% survival had no appreciable effects on u.v. survival and frequencies of spontaneous and u.v.-induced 6TGsup(r) mutation and SCE. However, reconstruction experiments revealed that pretreatments of 6TGsup(r) and 6TGsup(s) (wild-type) cells with these inhibitors for 6 days tended to block metabolic co-operation in their co-cultures. Thus, elastatinal and leupeptin are neither clastogenic nor mutagenic by themselves, and do not alter mutation fixation and expression. (author)

  8. Elastatinal and leupeptin: effects on u.v.-induced mutation and sister-chromatid exchanges in Chinese hamster cells

    International Nuclear Information System (INIS)

    Paul, P.; Fujiwara, Y.

    1981-01-01

    Microbial protease inhibitors elastatinal and leupeptin were tested for cytotoxicity and for effects on spontaneous and u.v.-induced 6-thioguanine-resistant (6TGr) mutation and sister-chromatid exchange (SCE) in V79 Chinese hamster cells. Continuous treatment with elastatinal exhibited marked cytotoxicity, while leupeptin was almost non-cytotoxic. Elastatinal rapidly induced cytotoxic effects as a function of its concentration and time of exposure. Near maximum cytotoxicity was reached after exposure of 6-8 h and this was partially abolished by the presence of 2.5 micrograms cycloheximide per ml. Concentrations of either protease inhibitor which gave 60-80% survival had no appreciable effects on u.v. survival and frequencies of spontaneous and u.v.-induced 6TGr mutation and SCE. However, reconstruction experiments revealed that pretreatments of 6TGr and 6TGs (wild-type) cells with these inhibitors for 6 days tended to block metabolic co-operation in their co-cultures. Thus, elastatinal and leupeptin are neither clastogenic mutagenic by themselves, and do not alter mutation fixation and expression

  9. DNA replication in ultraviolet light irradiated Chinese hamster cells: the nature of replicon inhibition and post-replication repair

    International Nuclear Information System (INIS)

    Doniger, J.

    1978-01-01

    DNA replication in ultraviolet light irradiated Chinese hamster cells was studied using techniques of DNA fiber autoradiography and alkaline sucrose sedimentation. Bidirectionally growing replicons were observed in the autoradiograms independent of the irradiation conditions. After a dose of 5 J/m 2 at 254 nm the rate of fork progression was the same as in unirradiated cells, while the rate of replication was reduced by 50%. After a dose of 10J/m 2 the rate of fork progression was reduced 40%, while the replication rate was only 25% of normal. Therefore, at low doses of ultraviolet light irradiation, the inhibition of DNA replication is due to reduction in the number of functioning replicons, while at higher doses the rate of fork progression is also slowed. Those replicons which no longer function after irradiation are blocked in fork movement rather than replicon initiation. After irradiation, pulse label was first incorporated into short nascent strands, the average size of which was approximately equal to the distance between pyrimidine dimers. Under conditions where post-replication repair occurs these short strands were eventually joined into larger pieces. Finally, the data show that slowing post-replication repair with caffeine does not slow fork movement. The results presented here support the post-replication repair model of 'gapped synthesis' and rule out a major role for 'replicative bypass'. (author)

  10. Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

    Science.gov (United States)

    Vita, N; Oury-Donat, F; Chalon, P; Guillemot, M; Kaghad, M; Bachy, A; Thurneyssen, O; Garcia, S; Poinot-Chazel, C; Casellas, P; Keane, P; Le Fur, G; Maffrand, J P; Soubrie, P; Caput, D; Ferrara, P

    1998-11-06

    The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

  11. Interaction between the effects of pepleomycin with lidocaine and radiation on cultured Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Shimada, Yuji

    1989-01-01

    The interaction of the cytotoxicities in combination use of lidocaine (LID), pepleomycin (PEP) and radiation in combined treatment was studied using Chinese hamster V79 cells by colony forming assay. LID showed no cytotoxic effect up to 12 mM when it was employed alone. However selective enhancement of the PEP cytotoxic effect appeared when the drugs were used simultaneously. The mechanism of this enhancing effect was thought to involve inhibition by LID of the repair of the cells from PEP potentially lethal damage. LID showed no enhancing effect on the radiation cytotoxicity when the agents were used simultaneously. Only an additive effect appeared when PEP and radiation were employed simultaneously. An interactive effect appeared when LID, PEP and radiation were used simultaneously, although this effect was not so significant. The enhancement ratio of PEP with LID on radiation was 1.58. The fundamental mechanism of enhancement of cytotoxic effect of LID on PEP and the interactive relationship among LID, PEP and radiation are analyzed and discussed. (author)

  12. Effects of copper oxide nanoparticles and copper ions to zebrafish (Danio rerio) cells, embryos and fry

    DEFF Research Database (Denmark)

    Thit, Amalie; Skjolding, Lars Michael; Selck, Henriette

    2017-01-01

    The use of engineered metal nanoparticles (NPs) is continuously increasing and so is the need for information regarding their toxicity. This study compares the toxicity of CuO NPs with ionic Cu in three zebrafish model systems; zebrafish hepatoma cell line (ZFL), fish embryo toxicity test (FET) a...

  13. Small Molecule Injection into Single-Cell C. elegans Embryos via Carbon-Reinforced Nanopipettes

    Science.gov (United States)

    Morton, Diane G.; Fellman, Shanna M.; Chung, SueYeon; Soltani, Mohammad; Kevek, Joshua W.; McEuen, Paul M.; Kemphues, Kenneth J.; Wang, Michelle D.

    2013-01-01

    The introduction of chemical inhibitors into living cells at specific times in development is a useful method for investigating the roles of specific proteins or cytoskeletal components in developmental processes. Some embryos, such as those of Caenorhabditis elegans, however, possess a tough eggshell that makes introducing drugs and other molecules into embryonic cells challenging. We have developed a procedure using carbon-reinforced nanopipettes (CRNPs) to deliver molecules into C. elegans embryos with high temporal control. The use of CRNPs allows for cellular manipulation to occur just subsequent to meiosis II with minimal damage to the embryo. We have used our technique to replicate classical experiments using latrunculin A to inhibit microfilaments and assess its effects on early polarity establishment. Our injections of latrunculin A confirm the necessity of microfilaments in establishing anterior-posterior polarity at this early stage, even when microtubules remain intact. Further, we find that latrunculin A treatment does not prevent association of PAR-2 or PAR-6 with the cell cortex. Our experiments demonstrate the application of carbon-reinforced nanopipettes to the study of one temporally-confined developmental event. The use of CRNPs to introduce molecules into the embryo should be applicable to investigations at later developmental stages as well as other cells with tough outer coverings. PMID:24086620

  14. The Effect of Prolonged Culture of Chromosomally Abnormal Human Embryos on The Rate of Diploid Cells

    Directory of Open Access Journals (Sweden)

    Masood Bazrgar

    2016-12-01

    Full Text Available Background: A decrease in aneuploidy rate following a prolonged co-culture of human blastocysts has been reported. As co-culture is not routinely used in assisted reproductive technology, the present study aimed to evaluate the effect of the prolonged single culture on the rate of diploid cells in human embryos with aneuploidies. Materials and Methods: In this cohort study, we used fluorescence in situ hybridization (FISH to reanalyze surplus blastocysts undergoing preimplantation genetic diagnosis (PGD on day 3 postfertilization. They were randomly studied on days 6 or 7 following fertilization. Results: Of the 30 analyzed blastocysts, mosaicism was observed in 26(86.6%, while 2(6.7% were diploid, and 2(6.7% were triploid. Of those with mosaicism, 23(88.5% were determined to be diploid-aneuploid and 3(11.5% were aneuploid mosaic. The total frequency of embryos with more than 50% diploid cells was 33.3% that was lower on day 7 in comparison with the related value on day 6 (P<0.05; however, there were no differences when the embryos were classified according to maternal age, blastocyst developmental stage, total cell number on day 3, and embryo quality. Conclusion: Although mosaicism is frequently observed in blastocysts, the prolonged single culture of blastocysts does not seem to increase the rate of normal cells.

  15. Identification of potential biomarkers in donor cows for in vitro embryo production by granulosa cell transcriptomics

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Salleh, Suraya M; Freude, Kristine

    2017-01-01

    The Ovum Pick Up-In vitro Production (OPU-IVP) of embryos is an advanced reproductive technology used in cattle production but the complex biological mechanisms behind IVP outcomes are not fully understood. In this study we sequenced RNA of granulosa cells collected from Holstein cows at oocyte...

  16. Activation of the ribosomal RNA genes late in the third cell cycle of porcine embryos

    DEFF Research Database (Denmark)

    Viuff, Dorthe; Greve, Torben; Holm, Peter

    2002-01-01

    ; there was no silver staining at the sites of the rRNA genes and nucleolus precursor bodies. From 30 hpc onwards, most 4-cell embryos had medium size to large clusters of FITC-labeled areas colocalized with silver staining of rRNA gene clusters and fibrillogranular nucleoli. These observations indicate that r...

  17. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    Science.gov (United States)

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  18. Differing sensitivity to fluorescent light in Chinese hamster cells containing equally incorporated quantities of BUdR versus IUdR

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Morstyn, G.; Russo, A.; Kinsella, T.J.; Fornace, A. Jr.; McPherson, S.; Glatstein, E.

    1984-01-01

    Chinese hamster V79 cells that had incorporated approximately equal levels of either BUdR or IUdR into their DNA were found to be equal sensitizers to x rays. However, BUdR-substituted cells were much more sensitive to fluorescent light than IUdR-substituted cells, both on a cell survival basis and by the initial number of single strand DNA breaks induced. Since a major toxicity to the use of BUdR clinically has been light-induced skin rash, these data indicate that the use of IUdR clinically might cause less untoward toxicity but yet provide the same radiosensitization as BUdR

  19. Replication of simian virus 40 in simian virus 40-transformed hamster kidney cells induced by mitomycin C or 60Co γ irradiation

    International Nuclear Information System (INIS)

    Rakusanova, T.; Smales, W.P.; Kaplan, J.C.; Black, P.H.

    1978-01-01

    Several clones of simian virus 40 (SV40)-transformed hamster kidney cells, which are heterogeneous for induction of infectious SV40, have been studied. SV40 yields are low after induction with 60 Co γ irradiation or mitomycin C. In order to clarify the mechanism(s) by which virus is produced in induced cells, we analyzed the replication of viral DNA and production of virion (V) antigen and infectious virus after induction in various clones as well as in lytically infected permissive cells. Cells replicating SV40 DNA or synthesizing V antigen were visualized by in situ hybridization and immunofluorescence techniques, respectively. Only some cells in induced cultures were found to produce SV40 and those which did were less efficient than lytically infected monkey cells. Mitomycin C or 60 Co γ irradiation acted by inducing more cells to replicate virus rather than by increasing the amount of SV40 released from individual cells. A greater proportion of cells could be induced to replicate SV40 DNA than to synthesize V antigen in all induced clones studied. Also, SV40 DNA replication was induced at lower doses of γ irradiation than the production of either V antigen or infectious virus suggesting that synthesis of late virus protein is more restricted in induced cells than is replication of SV40 DNA. These findings indicate that one of the effects of induction treatments on SV40-transformed hamster cells is an enhancement of the cells' capacity to support SV40 replication

  20. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    Energy Technology Data Exchange (ETDEWEB)

    Spigoti, Geyza; Bartolini, Paolo; Okazaki, Kayo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: kokazaki@ipen.br; Tsutsumi, Shiguetoshi [Amazon Food Ltd., Tokyo (Japan)], e-mail: fwip5138@mb.infoweb.ne.jp

    2009-07-01

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by {sup 60}Co {gamma}-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 {mu}g/ml), 1 h before irradiation, with 1 Gy of {gamma} radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  1. Protective effect of propolis on radiation-induced chromosomal damage on Chinese hamster ovary cells (CHO-K1)

    International Nuclear Information System (INIS)

    Spigoti, Geyza; Bartolini, Paolo; Okazaki, Kayo; Tsutsumi, Shiguetoshi

    2009-01-01

    In the last years, particular interest has been given to investigations concerning natural, effective and nontoxic compounds with radioprotective capacity in concert with increasing utilization of different types of ionizing radiation for various applications. Among them, propolis, a resinous mixture of substances collected by honey bees (Apis mellifera) has been considered promising since it presents several advantageous characteristics, i.e., antiinflammatory, anticarcinogenic, antimicrobial and free radical scavenging action. It is, therefore, a direct antioxidant that protects cells and organisms from the adverse effects of ionizing radiation. These relevant biological activities are mainly mediated by the flavonoids, present at relatively high concentrations in the propolis. Considering that the chemical composition and, consequently, the biological activity of propolis is variable according to the environmental plant ecology, the present study was conducted in order to evaluate the radioprotective capacity of Brazilian propolis, collected in the State of Rio Grande do Sul, against genotoxic damages induced by 60 Co γ-radiation in Chinese hamster ovary cells (CHO-K1). for this purpose, micronucleus induction was analyzed concerning irreparable damage, specifically related to DNA double-strand breaks, that are potentially carcinogenic. CHO-K1 cells were submitted to different concentrations of propolis (3 - 33 μg/ml), 1 h before irradiation, with 1 Gy of γ radiation (0.722 Gy/min). The data obtained showed a decreasing tendency in the quantity of radioinduced damage on cells previously treated with propolis. The radioprotective effect was more prominent at higher propolis concentration. The treatment with propolis alone did not induce genotoxic effects on CHO-K1 cells. Beside that, the treatment with propolis, associated or not with radiation, did not influence the kinetics of cellular proliferation. (author)

  2. Tripolar mitosis in human cells and embryos: occurrence, pathophysiology and medical implications.

    Science.gov (United States)

    Kalatova, Beata; Jesenska, Renata; Hlinka, Daniel; Dudas, Marek

    2015-01-01

    Tripolar mitosis is a specific case of cell division driven by typical molecular mechanisms of mitosis, but resulting in three daughter cells instead of the usual count of two. Other variants of multipolar mitosis show even more mitotic poles and are relatively rare. In nature, this phenomenon was frequently observed or suspected in multiple common cancers, infected cells, the placenta, and in early human embryos with impaired pregnancy-yielding potential. Artificial causes include radiation and various toxins. Here we combine several pieces of the most recent evidence for the existence of different types of multipolar mitosis in preimplantation embryos together with a detailed review of the literature. The related molecular and cellular mechanisms are discussed, including the regulation of centriole duplication, mitotic spindle biology, centromere functions, cell cycle checkpoints, mitotic autocorrection mechanisms, and the related complicating factors in healthy and affected cells, including post-mitotic cell-cell fusion often associated with multipolar cell division. Clinical relevance for oncology and embryo selection in assisted reproduction is also briefly discussed in this context. Copyright © 2014 Elsevier GmbH. All rights reserved.

  3. Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed

    International Nuclear Information System (INIS)

    Jegla, D.E.; Sussex, I.M.

    1989-01-01

    We mapped the fate of cells in the shoot meristem of the dry-seed embryo of sunflower, Helianthus annuus L. cv. Peredovic, using irradiation-induced somatic sectors. We analyzed 249 chlorophyll-deficient or glabrous (hairless) sectors generated in 236 plants. Most sectors observed in the inflorescence extended into vegetative nodes. Thus cell lineages that ultimately gave rise to reproductive structures also contributed to vegetative structures. No single sector extended the entire length of the shoot. Thus the shoot is not derived from one or a few apical initials. Rather, the position, vertical extent, and width of the sectors at different levels of the shoot suggest that the shoot is derived from three to four circumferential populations of cells in each of three cell layers of the embryo meristem. Sectors had no common boundaries even in plants with two or three independent sectors, but varied in extent and overlapped along the length of the shoot. Thus individual cells in a single circumferential population behaved independently to contribute lineages of different vertical extents to the growing shoot. The predicted number of circumferential populations of cells as well as the apparent cell number in each population was consistent with the actual number of cells in the embryo meristem observed in histological sections

  4. β-catenin functions pleiotropically in differentiation and tumorigenesis in mouse embryo-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Noriko Okumura

    Full Text Available The canonical Wnt/β-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. β-Catenin, encoded by the Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if β-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established β-catenin-deficient (β-cat(Δ/Δ mouse embryo-derived stem cells and showed that β-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs. However, teratomas formed from embryo-derived β-cat(Δ/Δ ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional β-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that β-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight β-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.

  5. Activation of mitochondrial promoter PH-binding protein in a radio-resistant Chinese hamster cell strain associated with Bcl-2

    International Nuclear Information System (INIS)

    Roychoudhury, Paromita; Ghosh, Utpal; Bhattacharyya, Nitai P.; Chaudhuri, Keya

    2006-01-01

    The cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P H ) has been increased in a radio-resistant cell strain designated as M5 in comparison with the parental Chinese hamster V79 cells. These genes are also up regulated in Chinese hamster V79 cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P L ) was found to be similar in both the cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial cell extracts from these two cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 cell extracts interacted with the P H promoter in comparison to that observed with cell extracts from Chinese hamster V79 cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 cells was due to enhanced interaction proteins with the promoters P H and mediated by the expression of Bcl2

  6. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2011-12-01

    Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

  7. Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

    Science.gov (United States)

    Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

    2008-03-01

    Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

  8. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

    Directory of Open Access Journals (Sweden)

    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  9. Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells.

    Science.gov (United States)

    Dreesen, Imke A J; Fussenegger, Martin

    2011-04-01

    Engineering of mammalian production cell lines to improve titer and quality of biopharmaceuticals is a top priority of the biopharmaceutical manufacturing industry providing protein therapeutics to patients worldwide. While many engineering strategies have been successful in the past decade they were often based on the over-expression of a single transgene and therefore limited to addressing a single bottleneck in the cell's production capacity. We provide evidence that ectopic expression of the global metabolic sensor and processing protein mammalian target of rapamycin (mTOR), simultaneously improves key bioprocess-relevant characteristics of Chinese hamster ovary (CHO) cell-derived production cell lines such as cell growth (increased cell size and protein content), proliferation (increased cell-cycle progression), viability (decreased apoptosis), robustness (decreased sensitivity to sub-optimal growth factor and oxygen supplies) and specific productivity of secreted human glycoproteins. Cultivation of mTOR-transgenic CHO-derived cell lines engineered for secretion of a therapeutic IgG resulted in antibody titers of up to 50 pg/cell/day, which represents a four-fold increase compared to the parental production cell line. mTOR-based engineering of mammalian production cell lines may therefore have a promising future in biopharmaceutical manufacturing of human therapeutic proteins. Copyright © 2010 Wiley Periodicals, Inc.

  10. Nicotinamide starvation and inhibition of poly(ADP-Ribose) synthesis enhance the induced mutation in Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Okada, Gensaku; Kaneko, Ichiro; Mitsui, Hideki.

    1987-01-01

    The effects of nicotinamide (NA) deficiency and added NA and 3-aminobenzamide (3AB) on the cytotoxicity and the induction of mutations in Chinese hamster V79-14 cells were investigated. In NA deficiency the addition of NA (up to 4 mM) and 3AB (up to 7.5 mM) was not cytotoxic. The presence of NA prior to exposure to mitomycin C (MMC) or γ-rays produced a dose-dependent increase in the relative cloning ability of DNA-damaged cells. The lethality of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was significantly potentiated by pre-treatment with 5 mM 3AB, but no potentiation by 3AB was observed for MMC, ultraviolet (UV)-B light, or γ-rays. Among cells pre-cultured in NA-free medium there were increased frequencies of mutations at both the hypoxanthineguanine phosphoribosyltransferase (HGPRT) and the adenine phosphoribosyltransferase (APRT) loci following DNA damage. The enhancing effect by NA deficiency was time-dependent. Incubation with NA prior to DNA damage produced a significant reduction in the frequency of mutations. The addition of 3AB to the nicotinamide adenine dinucleotide (NAD + )-depleted cell cultures before or after the DNA damage also strongly increased the frequency of induced mutations, with increasing concentrations of 3AB up to 5 mM, but the frequency was reduced at higher concentrations. The interaction between NA deficiency and the addition of 3AB appears to act synergistically on mutation induction. A correlation was observed between the potential of inhibiting poly (ADP-ribose) polymerase and the enhancement of mutation frequency. (author)

  11. The influence of the wavelength of ultraviolet radiation on survival, mutation induction and DNA repair in irradiated Chinese hamster cells

    International Nuclear Information System (INIS)

    Zelle, B.; Reynolds, R.J.; Kottenhagen, M.J.; Schuite, A.; Lohmann, P.H.M.

    1980-01-01

    Chinese hamster ovary cells were used to compare the cytotoxicity and mutagenicity of far-UV radiation emitted by a low-pressure mercury, germicidal lamp (wavelength predominantly 254 nm) with that of near UV radiation emitted by a fluorescent lamp with a continuous spectrum (Westinghouse Sun Lamp), of which only the radiation with wavelengths greater than 290 nm or greater than 310 nm was transmitted to the cells. The radiation effects were compared on the basis of an equal number of pyrimidine dimers, the predominant lesion induced in DNA by far-UV, for the induction of which much more energy is needed with near-UV than with 254-nm radiation. The numbers of dimers induced were determined by a biochemical method detecting UV-endonuclease-susceptible sites. The equivalence of these sites with pyrimidine dimers was established, qualitatively and quantitatively, in studies with enzymic photoreactivation in vitro and chromatographic analysis of dimers. On the basis of induced dimers, more cells were killed by UE 310-nm UV than by UE 290-nm UV; both forms of radiation were more cytotoxic than 254-nm UV when equal numbers of dimers were induced. Moreover, 5-6 times as many mutants were induced per dimer by UE 310-nm UV than by UE 290-nm UV; the latter appeared approximately as mutagenic as 254-nm UV. The differences in lethality and mutagenicity were not caused by differences in repair of dimers: cells with an equal number of dimers induced by either 254-nm or near-UV showed the same removal of sites susceptible to a UV endonuclease specific for dimers, as well as an identical amount of repair replication. The results indicate that near-UV induces, besides pyrimidine dimers, other lesions that appear to be of high biological significance. (orig.)

  12. Expression and activity levels of chymase in mast cells of burn wound tissues increase during the healing process in a hamster model.

    Science.gov (United States)

    Dong, Xianglin; Xu, Tao; Ma, Shaolin; Wen, Hao

    2015-06-01

    The present study aimed to investigate the changes in the expression levels and activity of mast cell chymase in the process of burn wound healing in a hamster model of deep second-degree burn. The hamster model was established by exposing a ~3 cm diameter area of bare skin to hot water (75°C) for 0, 6, 8, 10 or 12 sec. Tissue specimens were collected 24 h after burning and histological analysis revealed that hot water contact for 12 sec was required to produce a deep second-degree burn. Quantitative polymerase chain reaction and a radioimmunoassay were used to the determine changes in chymase mRNA expression levels and activity. The mRNA expression levels and activity of chymase were increased in the burn wound tissues when compared with the normal skin. However, no statistically significant differences were observed in mast cell chymase activity amongst the various post-burn stages. Chymase mRNA expression levels peaked at day 1 post-burn, subsequently decreasing at days 3 and 7 post-burn and finally increasing again at day 14 post-burn. In summary, a hamster model of deep second-degree burn can be created by bringing the skin into contact with water at 75°C for 12 sec. Furthermore, the mRNA expression levels and activity of chymase in the burn wound tissues increased when compared with those in normal skin tissues.

  13. Single-cell duplex RT-LATE-PCR reveals Oct4 and Xist RNA gradients in 8-cell embryos

    Directory of Open Access Journals (Sweden)

    Hartung Odelya

    2007-12-01

    Full Text Available Abstract Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when

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

    Science.gov (United States)

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

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

  15. Distinct functional characteristics of levocabastine sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

    Science.gov (United States)

    Yamada, M; Yamada, M; Lombet, A; Forgez, P; Rostène, W

    1998-01-01

    Neurotensin has been shown to produce pharmacological effects both in brain and periphery. Several of these effects are mediated by a high-affinity neurotensin NT1 receptor. On the other hand, a low-affinity levocabastine-sensitive neurotensin NT2 receptor was molecularly cloned from rodent brain recently. In this study, in contrast to NT1 receptor, levocabastine (a histamine H1 receptor antagonist) and SR48692 (an antagonist for NT1 receptor) strongly stimulated intracellular Ca2+ mobilization in transfected Chinese hamster ovary cells expressing rat NT2 receptor, thus acting as potent NT2 receptor. Furthermore, despite of their affinities for NT2 receptor, the Ca2+ responses to potent NT1 agonists, neurotensin or JMV449 ([Lys8-(CH2NH)-Lys9]Pro-Tyr-Ile-Leu, a peptidase resistant analogue of neurotensin) were much smaller than that observed with SR48692. These findings suggest that NT1 and NT2 receptors present distinct functional characteristics and that SR48692 may act as a potent agonist for NT2 receptor.

  16. Analysis of protein incorporation of radioactive isotopes in the Chinese hamster ovary cell cycle by electronic sorting and gel microelectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Pipkin, J.L.; Anson, J.F.; Hinson, W.G.; Schol, H.; Burns, E.R.; Casciano, D.A.

    1986-03-01

    The patterns of (3H)-leucine and (32P)-phosphate incorporation of proteins extracted with varying molarities of sodium chloride were analyzed from nuclei physically sorted from six fluorescence windows after propidium iodine staining of the G0 + G1 and G2 + M phases of the Chinese hamster ovary (CHO) cell cycle. Eight hundred nanograms of protein were used in each electrophoretic analysis obtained from 200,000 nuclei, a portion of the sample, from each window. Autoradiography was performed in a two-dimensional polyacrylamide gel ultra-microelectrophoresis apparatus (UMEA) designed and fabricated in this laboratory. There was a net reduction and/or loss of (3H)-leucine- and (32P)-phosphate-labeled protein regions from the autoradiographs occurring primarily in the G2 + M phase. Two phosphorylated proteins that were stage specific were observed in partitions of the G2 + M phase. The use of isolated proteins and the coelectrophoresis of these markers demonstrated the similarity in mobility of a number of proteins seen in the autoradiographs of proteins extracted with high and low salt molarities and implied they are synonymous. Coelectrophoresis indicated that a substantial number of high molecular weight proteins that decreased or disappeared at late stages of G2 + M and early mitosis were composed, in part, of nucleolar proteins.

  17. Human interleukin for DA cells or leukemia inhibitory factor is released by Vero cells in human embryo coculture.

    Science.gov (United States)

    Papaxanthos-Roche, A; Taupin, J L; Mayer, G; Daniel, J Y; Moreau, J F

    1994-09-01

    In the light of the newly discovered implications of human interleukin for DA cells and leukemia inhibitory factor in embryology, we searched for the presence of this soluble cytokine in the supernatant of Vero cell coculture systems. Using a bioassay as well as a specific ELISA, we demonstrated that Vero cells are able to release large quantities of human interleukin for DA cells and leukemia inhibitory factor in the embryo-growing medium of such cocultures.

  18. Sodium butyrate affects the cytotoxic and mutagenic response of V79 Chinese hamster cells to the genotoxic agents, daunorubicin and U.V. radiation

    International Nuclear Information System (INIS)

    Pani, B.; Babudri, N.; Giancotti, V.; Russo, E.

    1984-01-01

    It has been suggested that conditions which lead to modifications in the chromatin structure could be responsible for an increased accessibility of DNA to genotoxic agents in eukaryotic cells. With this in mind, the cytotoxic and mutagenic activity of the anthracycline antibiotic, daunorubicin, and of UV radiation was assayed on V79 Chinese hamster cells pretreated or not with 5 mM sodium butyrate, an agent known to induce modifications in the chromatin structure: this treatment in fact proved to induce the hyperacetylation of the core histones, and moreover to enhance the cytotoxic response of the cells to both daunorubicin and UV radiation and the mutagenic response to daunorubicin. (orig.)

  19. Effects of sphingosine-1-phosphate on gene expression of two cell mouse embryos induced by C2-Ceramide

    Directory of Open Access Journals (Sweden)

    Xujing Geng

    2014-06-01

    Conclusions: This study provides a map of genes in the pre-implantation two cell mouse embryo. Further investigation based on these data will provide a better understanding of the effects of S1P on the pre-implantation embryos in other mammalian species, especially human.

  20. Chinese hamster ovary (CHO-K1) cells expressed native insulin-like ...

    African Journals Online (AJOL)

    GREGORY

    2011-12-16

    Dec 16, 2011 ... ... University Malaysia (IIUM), P.O. Box 10, 50728, Kuala Lumpur, Malaysia. Accepted 7 November, 2011. Insulin-like growth factor-1 (IGF-1) has been shown to promote cell proliferation and inhibit apoptosis of cells. These are two characteristics of mammalian cell culture which may lead to high density cell.

  1. Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

    Directory of Open Access Journals (Sweden)

    Tong Zhang

    Full Text Available Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

  2. Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

    International Nuclear Information System (INIS)

    Zhong Zhisheng; Zhang Gang; Meng Xiaoqian; Zhang Yanling; Chen Dayuan; Schatten, Heide; Sun Qingyuan

    2005-01-01

    Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, γ-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. γ-tubulin translocated into the two poles of the transient spindles, while no accumulated γ-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, γ-tubulin was translocated to the spindle poles. The distribution of γ-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. γ-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as

  3. Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

    Science.gov (United States)

    Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C

    2014-02-01

    Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

  4. T cell precursor migration towards beta 2-microglobulin is involved in thymus colonization of chicken embryos

    DEFF Research Database (Denmark)

    Dunon, D; Kaufman, J; Salomonsen, J

    1990-01-01

    beta 2-microglobulin (beta 2m) attracts hemopoietic precursors from chicken bone marrow cells in vitro. The cell population responding to beta 2m increases during the second period of thymus colonization, which takes place at days 12-14 of incubation. The precursors from 13.5 day old embryos were...... isolated after migration towards beta 2m in vitro and shown to be able to colonize a 13 day old thymus in ovo, where they subsequently acquire thymocyte markers. In contrast these beta 2m responsive precursors did not colonize embryonic bursa, i.e. differentiate into B lymphocytes. During chicken...... embryogenesis, peaks of beta 2m transcripts and of free beta 2m synthesis can only be detected in the thymus. The peak of free beta 2m synthesis in the thymus and the increase of beta 2m responding bone marrow cells both occur concomitantly with the second wave of thymus colonization in chicken embryo, facts...

  5. [Observation in situ of differentiation from PGC to hematopoietic system cells in chicken embryo].

    Science.gov (United States)

    Zhou, Dong-Yu; Liu, Rong-Xiu; Pei, Yue-Hu

    2009-02-01

    To study the relationship between hematopoiesis and primordial germ cells, chick embryos at different developing stages were flatbed and located. After fixed by glutaral, the embryos were PAS and HE stained respectively, dehydrated serially, transparent, mounted, and were observed in situ or in cut sheet condition. The results showed: (1) the cellule amorphous and the disposition in chick embryo of PGCs were coincident no matter stained by PAS or HE staining, and HE staining could disclose the morphologic characteristics more clearly, exactly and completely; (2) genesis of blood island could be observed at the boundary of light and dark region of the extraembryonic blastoderm at about 26 hours; (3) both the blood vessel endothelium cells and free cells of the blood island were differentiated from PGCs. The generating of genuine yolk sac was at about 44 - 48 hours. It is concluded that the initial anatomic site of blood island genesis may be is mesoblast of extraembryonic blastoderm rather than the yolk sac; the blood vessel endothelium cells and the blood cells are generated parallel; the PGCs are the common ancestry of angioblast and HSC.

  6. Chinese hamster ovary (CHO-K1) cells expressed native insulin-like ...

    African Journals Online (AJOL)

    These are two characteristics of mammalian cell culture which may lead to high density cell culture producing optimal desired yield of bioproducts. An inherent secretion of IGF-1 protein from host cells into the culture media is hypothesized to enable reduction or removable of serum from culture media, thus reducing cost.

  7. Chemoprevention by Quercetin of Oral Squamous Cell Carcinoma by Suppression of the NF-κB Signaling Pathway in DMBA-treated Hamsters.

    Science.gov (United States)

    Zhang, Wen; Yin, Gang; Dai, Jianguo; Sun, Y U; Hoffman, Robert M; Yang, Zhijian; Fan, Yuan

    2017-08-01

    The aim of this study was to investigate the effects of the flavonoid quercetin on chemoprevention of oral squamous cell carcinoma (OSCC). The study involved molecular signaling pathways in 7,12-dimethylbenz(a) anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. DMBA (0.5%) was painted at the right buccal pouches of hamsters for 14 weeks to induce carcinoma. DMBA-treated hamsters received simultaneous doses of quercetin. Animals without DMBA induction were used as normal controls. The incidence of OSCC and the severity of pre-malignant lesions were determined histologically. Apoptosis in the pouch tissue was determined by TUNEL staining. The mRNA and protein expression of NF-κB p50 and p65, as well as Bcl-2 and Bax genes were analyzed using RT-PCR and Western blotting, respectively. Quercetin, at various doses, significantly reduced OSCC incidence and severity of hyperplasia and dysplasia compared to the DMBA-induction-only group (p<0.01). Apoptosis was induced by quercetin treatment compared to the DMBA-induction-only group (p<0.01). mRNA and protein expression of NF-κB p50, p65 as well as Bcl-2 genes were significantly suppressed by quercetin at high doses compared to DMBA induction only (p<0.05). However, mRNA and protein expression of the Bax gene was increased by quercetin treatment at medium and high doses, compared to the DMBA-induction-only group (p<0.05). Quercetin significantly reduced body-weight loss compared to the DMBA-induction-only group (p<0.05). Quercetin reduced tumor incidence and induced apoptosis through modulation of NF-κB signaling and its target genes Bcl-2 and Bax in the DMBA-induced carcigenesis hamster model, suggesting the potential of quercetin as a candidate for OSCC chemoprevention. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins.

    Directory of Open Access Journals (Sweden)

    Teruhiko Suzuki

    Full Text Available Microcell-mediated chromosome transfer (MMCT is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell.

  9. In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

    Science.gov (United States)

    Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

    2010-02-18

    The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

  10. Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Kuo, W.L.; Meyn, R.E.; Haidle, C.W.

    1984-01-01

    The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity

  11. Primordial germ cells and amnion development in the avian embryo

    NARCIS (Netherlands)

    De Melo Bernardo, Ana

    2016-01-01

    Primordial germ cells (PGCs) are the progenitors of the gametes, responsible for transmitting genetic information from generation to generation. Although there is a long history of gamete biology research, there is still a lot to be learned about many of the mechanisms underlying germ cell

  12. Effects of 3-Deoxyadenosine (Cordycepin) on the repair of X-ray-induced DNA single- and double-strand breaks in chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Hiraoka, Wakako; Kuwabara, Mikinori; Sato, Fumiaki

    1990-01-01

    The ability of cordycepin to inhibit the repair of DNA strand breaks was examined with X-irradiated Chinese hamster V79 cells in log-phase culture. A filter elution technique revealed that 70 μM cordycepin did not inhibit the repair of single-strand breaks but inhibited the repair of double-strand breaks. These findings confirmed the fact that the increase in the lethality of cordycepin in X-irradiated cultured mammalian cells was attributable to unrepaired DNA double-strand breaks. (author)

  13. Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

    Directory of Open Access Journals (Sweden)

    LiBing Ma

    Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

  14. Improved gene amplification by cell-cycle engineering combined with the Cre-loxP system in Chinese hamster ovary cells.

    Science.gov (United States)

    Matsuyama, Rima; Tsutsui, Tomomi; Lee, Kyoung Ho; Onitsuka, Masayoshi; Omasa, Takeshi

    2015-12-01

    The dihydrofolate reductase gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the industrial production of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  15. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    Directory of Open Access Journals (Sweden)

    Ingrid R. Cordeiro

    2015-09-01

    Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

  16. Canine distemper virus utilizes different receptors to infect chicken embryo fibroblasts and vero cells.

    Science.gov (United States)

    Chen, Jun; Liang, Xiu; Chen, Pei-fu

    2011-04-01

    Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts (CEF) is a common method to develop attenuated live vaccines with full security. Canine distemper virus (CDV) also does this, but the mechanisms and particular receptors remain unclear. Virus overlay protein blot assays were carried out on CEF membrane proteins, which were extracted respectively with a Mem-PER™ kit, a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method, and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells, indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.

  17. Effects of hyperthermia on growth kinetics of Chinese hamster ovarian carcinoma cells

    International Nuclear Information System (INIS)

    Leeper, D.B.; Bobyock, S.B.

    1987-01-01

    The effects of hyperthermia on growth rate, cell volume, and density at plateau phase were studied in OvCa cells in monolayer culture in McCoy's 5a + 10% FCS. At 37 0 C, T/sub G/=9.3 hr, cell density at plateau was 32 x 10/sup 4//cm/sup 2/, and mean cell volume decreased from 1200 μ/sup 3/ at the onset of exponential growth to 850 μ/sup 3/ in plateau phase. Cells were acutely heated for 60' at 43 0 ,30' at 44 0 , or 15' at 45 0 (S.F.=20%) and incubated at 37 0 ; or were chronically heated for up to 80 hr at 39-42 0 . Acute heating at 43-45 0 delayed cell division for appx 13 hr after which growth resumed with a T/sub G/=18 hr. Incubation at 39-40 0 had no effect on T/sub G/, but temperatures of 40.5-42 0 increased T/sub G/ at ΔH=176 kcal/mole. Increasing incubation temperature decreased cell density at plateau phase and altered cell volume kinetics. Cell density in plateau phase was 20 x 10/sup 4//cm/sup 2/ at 39 0 , 13 x 10/sup 4//cm/sup 2/ at 40 0 , 5x10/sup 4//cm/sup 2/ at 41 0 . Growth was greatly reduced at 42 0 (T/sub G/=55 hr) and doubling did not occur before onset of cell lysis. The decrease in cell volume with growth of the culture was unaffected at 39 0 . However, at temperatures ≥40 0 cell volume transiently increase, and the rate of decrease in volume that normally occurred with growth at 37-39 0 was less such that at 41 0 there was no decrease in volume at all before cells entered plateau phase. The authors' hypothesis is that the effects of heat on growth kinetics are related to alterations in rates of protein synthesis. This is currently being tested

  18. Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

    Directory of Open Access Journals (Sweden)

    Page Grier P

    2009-04-01

    Full Text Available Abstract Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT. Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively. However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research.

  19. Single-Cell Profiling of Epigenetic Modifiers Identifies PRDM14 as an Inducer of Cell Fate in the Mammalian Embryo

    Directory of Open Access Journals (Sweden)

    Adam Burton

    2013-11-01

    Full Text Available Cell plasticity or potency is necessary for the formation of multiple cell types. The mechanisms underlying this plasticity are largely unknown. Preimplantation mouse embryos undergo drastic changes in cellular potency, starting with the totipotent zygote through to the formation of the pluripotent inner cell mass (ICM and differentiated trophectoderm in the blastocyst. Here, we set out to identify and functionally characterize chromatin modifiers that define the transitions of potency and cell fate in the mouse embryo. Using a quantitative microfluidics approach in single cells, we show that developmental transitions are marked by distinctive combinatorial profiles of epigenetic modifiers. Pluripotent cells of the ICM are distinct from their differentiated trophectoderm counterparts. We show that PRDM14 is heterogeneously expressed in 4-cell-stage embryos. Forced expression of PRDM14 at the 2-cell stage leads to increased H3R26me2 and can induce a pluripotent ICM fate. Our results shed light on the epigenetic networks that govern cellular potency and identity in vivo.

  20. A preliminary investigation into the extent of increased radioresistance or hyper-radiosensitivity in cells of hamster cell lines known to be deficient in DNA repair

    International Nuclear Information System (INIS)

    Skov, K.; Marples, B.; Matthews, J.B.; Zhou, H.; Joiner, M.C.

    1994-01-01

    The response to low doses of X rays was assessed in cells of three hamster cell lines which are defective in DNA repair and was compared with their parental lines. Cells of the V79-derived double-strand break repair-deficient line XR-V15B showed no radioresistance in the 0.5-Gy range compared with the V79B wild type, but instead showed an exponential response. Cells of the single-strand break repair-deficient line EM9 showed hyper-radiosensitivity and exhibited increased radioresistance. Most interestingly, cells of the UV-20 cell line appeared to respond exponentially, as a continuation of the hyper-radiosensitive portion of the curve, with no evidence of increased radioresistance. This line is defective in an incision step of excision repair and is sensitive to crosslinking agents. Further studies are warranted to address the possible role of single- and double-strand break repair and excision repair in hyper-radiosensitivity and increased radioresistance. 24 refs., 4 figs

  1. Growth of primary embryo cells in a microculture system.

    Science.gov (United States)

    Villa, Max; Pope, Sara; Conover, Joanne; Fan, Tai-Hsi

    2010-04-01

    We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 microm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.

  2. Hydroxyurea does not prevent synchronized G1 Chinese hamster cells from entering the DNA synthetic period

    International Nuclear Information System (INIS)

    Walters, R.A.; Tobey, R.A.; Hildebrand, C.E.

    1976-01-01

    Using very high concentrations of radioactively labeled thymidine, we show that synchronized G 1 cells treated with hydroxyurea entered the DNA synthetic period at a time and rate indistinguishable from that of untreated cells, although the rate of DNA synthesis was greatly reduced in the drug-treated cultures. The DNA synthesized in the presence of hydroxyurea was less than or equal to 1 x 10 7 daltons, all of which could be chased into bulk DNA of approximately 3.5 x 10 8 daltons within 3 hr after removal of hydroxyurea. Hydroxyurea synchronized cells are apparently not blocked at the G 1 /S boundary but in the S phase itself

  3. DOT1L inhibitor improves early development of porcine somatic cell nuclear transfer embryos

    DEFF Research Database (Denmark)

    Tao, Jia; Zhang, Yu; Zuo, Xiaoyuan

    2017-01-01

    Incomplete epigenetic reprogramming of the genome of donor cells causes poor early and full-term developmental efficiency of somatic cell nuclear transfer (SCNT) embryos. Previous research indicate that inhibition of the histone H3 K79 methyltransferase DOT1L, using a selective pharmacological...... inhibitor EPZ004777 (EPZ), significantly improved reprogramming efficiency during the generation of mouse induced pluripotent stem cells. However, the roles of DOT1L in porcine nuclear transfer-mediated cellular reprogramming are not yet known. Here we showed that DOT1L inhibition via 0.5 nM EPZ treatment...

  4. Interactions of adriamycin and X-rays in Chinese hamster cells

    International Nuclear Information System (INIS)

    Meder, G.

    1982-01-01

    The effect of a combined ADM-and-radiation treatment of varying intervals was investigated in vitro. ADM sensitized the cells for the subsequent irradiation. This action was noticeable after 7h as a synergystic and after 2 and 3d as an additive effect of the combination treatment. Obviously, the ADM damage was inherited by the following cell generations. After 7d the sensitization of the cells could no longer be proved. From these results and those of other authors some general conclusions can be drawn. Different cell types react differently to the same kind of treatment. All forms of interaction are noticeable. Moreover, the ADM damage is obviously inherited. It must be assumed that this variability of the phenomena observed in vitro is also found in the human organism. (orig.) [de

  5. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Science.gov (United States)

    Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

    2012-01-01

    Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

  6. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Directory of Open Access Journals (Sweden)

    Juliana Branco Novo

    2012-01-01

    Full Text Available Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr− cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa and secreted (63–69 kDa form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

  7. The first cell-fate decisions in the mouse embryo: destiny is a matter of both chance and choice.

    Science.gov (United States)

    Zernicka-Goetz, Magdalena

    2006-08-01

    Development of the early mouse embryo has always been classified as regulative, meaning that when parts or blastomeres of the embryo are isolated they change their developmental fate and can even reconstruct the whole. However, regulative development does not mean that, in situ, these parts or blastomeres are equivalent; it does not mean that the early mammalian embryo is a ball of identical cells without any bias. Regulative development simply means that whatever bias the regions of the embryo might have they still remain flexible and can respond to experimental interference by changes of fate. This realization -- that regulative development and patterning can co-exist -- has led to a renaissance of interest in the first days of development of the mouse embryo, and several laboratories have provided evidence for some early bias. Now the challenge is to gain some understanding of the molecular basis of this bias.

  8. Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice.

    Science.gov (United States)

    Tarkowski, Andrzej K; Suwińska, Aneta; Czołowska, Renata; Ożdżeński, Wacław

    2010-12-15

    Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Effects of 5 Thio-D-Glucose on cellular adenosine triphosphate levels and deoxyribonucleic acid rejoining in hypoxic and aerobic Chinese hamster cells

    International Nuclear Information System (INIS)

    Nagle, W.A.; Moss, A.J. Jr.; Roberts, H.G. Jr.; Baker, M.L.

    1980-01-01

    Intracellular adenosine triphosphate (ATP) levels were measured in both hypoxic and aerobic cultures of V79 Chinese hamster cells treated with 5-thio-D-glucose (5-SH-D-Glc). This glucose analog, a known inhibitor of D-glucose transport and metabolism, reduced ATP in cell cultures allowed to become hypoxic by cell metabolism, but not in aerobic cultures treated similarly. Cells depleted of ATP were unable to rejoin x-ray induced deoxyribonucleic acid (DNA) strand breaks as measured by the alkaline sucrose gradient sedimentation technique. The inference for radiation therapy is that inhibition of glucose metabolism selectively depletes energy reserves in hypoxic cells, rendering these cells more radiosensitive and leading to a more effective tumor treatment

  10. Co-induction of glucose regulated proteins and adriamycin resistance in Chinese hamster cells

    International Nuclear Information System (INIS)

    Shen, J.; Hughes, C.; Cai, J.; Bartels, C.; Gessner, T.; Subjeck, J.

    1987-01-01

    Glucose deprivation, anoxia, calcium ionophore A23187 or 2-deoxyglucose all inducers of glucose regulated proteins (grps), also lead to a significant induction of resistance to the drug adriamycin. In the case of anoxia, A23187 and 2-deoxyglucose, the induction of resistance correlates with both the application of the inducing stress and the induction of grps. In the case of glucose deprivation, the onset of resistance correlates with the onset of glucose deprivation and precedes grp induction. Removal of each grp including condition results in the rapid disappearance of this resistance in a manner which correlates with the repression of the grps. This drug resistance can be induced in confluent cells or in actively proliferating cells, although the effect is greater in the more sensitive proliferating cells. Induction of heat shock proteins (hsps) does not appear to lead to any major change in adriamycin resistance. Grp induced cells retain less adriamycin than do controls with the greatest reduction occurring during anoxia, which is also the strongest inducer of grps and resistance. The authors propose that the application of a grp inducing stress leads to a concurrent induction in drug resistance, possibly via the translocation of grps in the cell. Finally, they also observed that adriamycin itself can induce both hsps and grps. It is possible that adriamycin exposure may correspondingly induce auto-resistance

  11. Pluripotency maintenance in mouse somatic cell nuclear transfer embryos and its improvement by treatment with the histone deacetylase inhibitor TSA.

    Science.gov (United States)

    Hai, Tang; Hao, Jie; Wang, Liu; Jouneau, Alice; Zhou, Qi

    2011-02-01

    Reprogramming of somatic cells to pluripotency can be achieved by nuclear transfer into enucleated oocytes (SCNT). A key event of this process is the demethylation of the Oct4 gene and its temporally and spatially regulated expression. Different studies have shown that it occurs abnormally in some SCNT embryos. TSA is a histone deacetylase inhibitor known to increase the efficiency of development to term of SCNT embryos, but its impact on the developmental features of SCNT embryos is poorly understood. Here, we have followed the fate of the pluripotent cells within SCNT embryos, from the late blastocyst to the early epiblast prior to gastrulation. Our data show a delay in development correlated with a defect in forming and maintaining a correct number of Oct4 expressing ICM and epiblast cells in SCNT embryos. As a consequence, during the outgrowth phase of embryonic stem cell derivation as well as during diapause in vivo, part of the SCNT blastocysts completely lose their ICM cells. Meanwhile, the others display a correctly reprogrammed ICM compatible with the derivation of ES cells and development of the epiblast. Our data also indicate that TSA favors the establishment of pluripotency in SCNT embryos.

  12. Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth

    Directory of Open Access Journals (Sweden)

    Alexander B. Opoku-Acheampong

    2016-01-01

    Full Text Available Saw palmetto supplements (SPS are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n=3; p<0.05. Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n=4, HLLP, HLHP, and HMLP SPS (n=6 groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p<0.05. Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth.

  13. Effect of Saw Palmetto Supplements on Androgen-Sensitive LNCaP Human Prostate Cancer Cell Number and Syrian Hamster Flank Organ Growth.

    Science.gov (United States)

    Opoku-Acheampong, Alexander B; Penugonda, Kavitha; Lindshield, Brian L

    2016-01-01

    Saw palmetto supplements (SPS) are commonly consumed by men with prostate cancer. We investigated whether SPS fatty acids and phytosterols concentrations determine their growth-inhibitory action in androgen-sensitive LNCaP cells and hamster flank organs. High long-chain fatty acids-low phytosterols (HLLP) SPS ≥ 750 nM with testosterone significantly increased and ≥500 nM with dihydrotestosterone significantly decreased LNCaP cell number. High long-chain fatty acids-high phytosterols (HLHP) SPS ≥ 500 nM with dihydrotestosterone and high medium-chain fatty acids-low phytosterols (HMLP) SPS ≥ 750 nM or with androgens significantly decreased LNCaP cell number (n = 3; p < 0.05). Five- to six-week-old, castrated male Syrian hamsters were randomized to control (n = 4), HLLP, HLHP, and HMLP SPS (n = 6) groups. Testosterone or dihydrotestosterone was applied topically daily for 21 days to the right flank organ; the left flank organ was treated with ethanol and served as the control. Thirty minutes later, SPS or ethanol was applied to each flank organ in treatment and control groups, respectively. SPS treatments caused a notable but nonsignificant reduction in the difference between left and right flank organ growth in testosterone-treated SPS groups compared to the control. The same level of inhibition was not seen in dihydrotestosterone-treated SPS groups (p < 0.05). Results may suggest that SPS inhibit 5α-reductase thereby preventing hamster flank organ growth.

  14. Role of ooplasm in nuclear and nucleolar remodeling of intergeneric somatic cell nuclear transfer embryos during the first cell cycle

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, Frantisek; Petrovicova, Ida

    2011-01-01

    Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-c...... in sequence-specific interactions between the ooplasm and chromatin of another genus. In conclusion, the results demonstrate a possible reason why the intergeneric SCNT embryos never reached the full term....

  15. Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos

    Directory of Open Access Journals (Sweden)

    S.F. Avram

    2013-01-01

    Full Text Available The P1.HTR cell line includes highly transfectable cells derived from P815 mastocytoma cells originating from mouse breast tissue. Despite its widespread use in immunogenic studies, no data are available about the behavior of P1.HTR cells in the chick embryo chorioallantoic membrane model. The objective of the present investigation was to study the effects of P1.HTR cells implanted on the chorioallantoic membrane of chick embryos. We inoculated P1.HTR cells into the previously prepared chick embryo chorioallantoic membrane and observed the early and late effects of these cells by stereomicroscopy, histochemistry and immunohistochemistry. A highly angiotropic and angiogenic effect occurred early after inoculation and a tumorigenic potential with the development of mastocytoma keeping well mast cells immunophenotype was detected later during the development. The P1.HTR mastocytoma cell line is a good tool for the development of the chick embryo chorioallantoic membrane mastocytoma model and also for other studies concerning the involvement of blood vessels. The chick embryo chorioallantoic membrane model of mastocytoma retains the mast cell immunophenotype under experimental conditions and could be used as an experimental tool for in vivo preliminary testing of antitumor and antivascular drugs.

  16. Organ and Tissue-Specific Localisation of Selected Cell Wall Epitopes in the Zygotic Embryo of Brachypodium distachyon

    Directory of Open Access Journals (Sweden)

    Alexander Betekhtin

    2018-03-01

    Full Text Available The plant cell wall shows a great diversity regarding its chemical composition, which may vary significantly even during different developmental stages. In this study, we analysed the distribution of several cell wall epitopes in embryos of Brachypodium distachyon (Brachypodium. We also described the variations in the nucleus shape and the number of nucleoli that occurred in some embryo cells. The use of transmission electron microscopy, and histological and immunolocalisation techniques permitted the distribution of selected arabinogalactan proteins, extensins, pectins, and hemicelluloses on the embryo surface, internal cell compartments, and in the context of the cell wall ultrastructure to be demonstrated. We revealed that the majority of arabinogalactan proteins and extensins were distributed on the cell surface and that pectins were the main component of the seed coat and other parts, such as the mesocotyl cell walls and the radicula. Hemicelluloses were localised in the cell wall and outside of the radicula protodermis, respectively. The specific arrangement of those components may indicate their significance during embryo development and seed germination, thus suggesting the importance of their protective functions. Despite the differences in the cell wall composition, we found that some of the antibodies can be used as markers to identify specific cells and the parts of the developing Brachypodium embryo.

  17. X-ray-sensitive mutants of Chinese hamster ovary cell line

    International Nuclear Information System (INIS)

    Jeggo, P.A.; Kemp, L.M.

    1983-01-01

    A standard technique of microbial genetics, which involves the transfer of cells from single colonies by means of sterile toothpicks, has been adapted to somatic cell genetics. Its use has been demonstrated in the isolation of X-ray-sensitive mutants of CHO cells. 9000 colonies have been tested and 6 appreciably X-ray-sensitive mutants were isolated. (D 10 values 5-10-fold of wild-type D 10 value.) A further 6 mutants were obtained which showed a slight level of sensitivity (D 10 values less than 2-fold of wild-type D 10 value). The 6 more sensitive mutants were also sensitive to bleomycin, a chemotherapeutic agent inducing X-ray-like damage. Cross-sensitivity to UV-irradiation and treatment with the alkylating agents, MMS, EMS and MNNG, was investigated for these mutants. Some sensitivity to these other agents was observed, but in all cases it was less severe than the level of sensitivity to X-irradiation. Each mutant showed a different overall response to the spectrum of agents examined and these appear to represent new mutant phenotypes derived from cultured mammalian cell lines. One mutant strain, xrs-7, was cross-sensitive to all the DNA-damaging agents, but was proficient in the repair of single-strand breaks. (Auth.)

  18. Trends and approaches in N-Glycosylation engineering in Chinese hamster ovary cell culture

    DEFF Research Database (Denmark)

    Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    will summarize a group of recent strategies andapproaches and come up with case studies for N-glycosylation engineering in CHO cells and show several examples of relevantstudy cases from our research: 1) media and feed design, 2) culture process optimization, 3) substrate addition, 4) geneticengineering, 5...

  19. The Chromatin Regulator Brpf1 Regulates Embryo Development and Cell Proliferation*

    Science.gov (United States)

    You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs. PMID:25773539

  20. Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

    Science.gov (United States)

    Wang, Chao-Jie; Wang, Guang; Wang, Xiao-Yu; Liu, Meng; Chuai, Manli; Lee, Kenneth Ka Ho; He, Xiao-Song; Lu, Da-Xiang; Yang, Xuesong

    2016-06-15

    Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

  1. Cell lineage of timed cohorts of Tbx6-expressing cells in wild-type and Tbx6 mutant embryos

    Directory of Open Access Journals (Sweden)

    Daniel Concepcion

    2017-07-01

    Full Text Available Tbx6 is a T-box transcription factor with multiple roles in embryonic development as evidenced by dramatic effects on mesoderm cell fate determination, left/right axis determination, and somite segmentation in mutant mice. The expression of Tbx6 is restricted to the primitive streak and presomitic mesoderm, but some of the phenotypic features of mutants are not easily explained by this expression pattern. We have used genetically-inducible fate mapping to trace the fate of Tbx6-expressing cells in wild-type and mutant embryos to explain some of the puzzling features of the mutant phenotype. We created an inducible Tbx6-creERT2 transgenic mouse in which cre expression closely recapitulates endogenous Tbx6 expression both temporally and spatially. Using a lacZ-based Cre reporter and timed tamoxifen injections, we followed temporally overlapping cohorts of cells that had expressed Tbx6 and found contributions to virtually all mesodermally-derived embryonic structures as well as the extraembryonic allantois. Contribution to the endothelium of major blood vessels may account for the embryonic death of homozygous mutant embryos. In mutant embryos, Tbx6-creERT2-traced cells contributed to the abnormally segmented anterior somites and formed the characteristic ectopic neural tubes. Retention of cells in the mutant tail bud indicates a deficiency in migratory behavior of the mutant cells and the presence of Tbx6-creERT2-traced cells in the notochord, a node derivative provides a possible explanation for the heterotaxia seen in mutant embryos.

  2. Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines

    International Nuclear Information System (INIS)

    Bodewein, Lambert; Schmelter, Frank; Di Fiore, Stefano; Hollert, Henner; Fischer, Rainer; Fenske, Martina

    2016-01-01

    Dendrimers are an emerging class of polymeric nanoparticles with beneficial biomedical applications like early diagnostics, in vitro gene transfection or controlled drug delivery. However, the potential toxic impact of exposure on human health or the environment is often inadequately defined. Thus, polyamidoamine (PAMAM) dendrimers of generations G3.0, 3.5, 4.0, 4.5 and 5.0 and polypropylenimine (PPI) dendrimers G3.0, 4.0 and 5.0 were tested in zebrafish embryos for 96 h and human cancer cell lines for 24 h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, with EC50 values ranging from 0.16 to just below 1.7 μM at 24 and 48 hpf. The predominant effects were mortality, plus reduced heartbeat and blood circulation for PPI dendrimers. Apoptosis in the embryos increased in line with the general toxicity concentration-dependently. Hatch and dechorionation of the embryos increased the toxicity, suggesting a protective role of the chorion. Lower generation dendrimers were more toxic in the embryos whereas the toxicity in the HepG2 and DU145 cell lines increased with increasing generation of cationic PAMAMs and PPI dendrimers. HepG2 were less sensitive than DU145 cells, with IC50 values ≥ 402 μM (PAMAMs) and ≤ 240 μM (PPIs) for HepG2 and ≤ 13.24 μM (PAMAMs) and ≤ 12.84 μM (PPIs) for DU145. Neither in fish embryos nor cells toxicity thresholds were determinable for anionic PAMAM G3.5 and G4.5. The study demonstrated that the cytotoxicity underestimated the in-vivo toxicity of the dendrimers in the fish embryos. - Highlights: • Zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time. • Zebrafish embryo toxicity of cationic dendrimers did not increase with generation. • Cationic dendrimers induced apoptosis in zebrafish embryos. • Toxicity of cationic dendrimers was lower in HepG2 and DU145 than zebrafish embryos.

  3. Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Bodewein, Lambert [Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Frankfurt & Aachen (Germany); Institute of Environmental Research (Biology V), RWTH Aachen University (Germany); Schmelter, Frank; Di Fiore, Stefano [Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Molecular Biology Division, Aachen (Germany); Hollert, Henner [Institute of Environmental Research (Biology V), RWTH Aachen University (Germany); Fischer, Rainer [Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Frankfurt & Aachen (Germany); Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Molecular Biology Division, Aachen (Germany); Fenske, Martina, E-mail: martina.fenske@ime.fraunhofer.de [Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Frankfurt & Aachen (Germany)

    2016-08-15

    Dendrimers are an emerging class of polymeric nanoparticles with beneficial biomedical applications like early diagnostics, in vitro gene transfection or controlled drug delivery. However, the potential toxic impact of exposure on human health or the environment is often inadequately defined. Thus, polyamidoamine (PAMAM) dendrimers of generations G3.0, 3.5, 4.0, 4.5 and 5.0 and polypropylenimine (PPI) dendrimers G3.0, 4.0 and 5.0 were tested in zebrafish embryos for 96 h and human cancer cell lines for 24 h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, with EC50 values ranging from 0.16 to just below 1.7 μM at 24 and 48 hpf. The predominant effects were mortality, plus reduced heartbeat and blood circulation for PPI dendrimers. Apoptosis in the embryos increased in line with the general toxicity concentration-dependently. Hatch and dechorionation of the embryos increased the toxicity, suggesting a protective role of the chorion. Lower generation dendrimers were more toxic in the embryos whereas the toxicity in the HepG2 and DU145 cell lines increased with increasing generation of cationic PAMAMs and PPI dendrimers. HepG2 were less sensitive than DU145 cells, with IC50 values ≥ 402 μM (PAMAMs) and ≤ 240 μM (PPIs) for HepG2 and ≤ 13.24 μM (PAMAMs) and ≤ 12.84 μM (PPIs) for DU145. Neither in fish embryos nor cells toxicity thresholds were determinable for anionic PAMAM G3.5 and G4.5. The study demonstrated that the cytotoxicity underestimated the in-vivo toxicity of the dendrimers in the fish embryos. - Highlights: • Zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time. • Zebrafish embryo toxicity of cationic dendrimers did not increase with generation. • Cationic dendrimers induced apoptosis in zebrafish embryos. • Toxicity of cationic dendrimers was lower in HepG2 and DU145 than zebrafish embryos.

  4. Cell survival after the combined action of manganese (MnCl2) and X-rays in synchronized Chinese hamster cells

    International Nuclear Information System (INIS)

    Skreb, Y.; Nagy, B.

    1984-01-01

    The interactions between the effects of manganese chloride and X-rays were studied in synchronized populations of V79 Chinese hamster fibroblasts. The cells were selected by shaking off asynchronous cultures for detachment of mitotic cells which were plated in petri dishes and exposed to various treatments. Irradiation was carried out with a Philips RT-100 X-ray unit. A final concentration of 0.25 mM MnCl 2 was used. The main parameter was the colony forming ability of the surviving cell fraction. When MnCl 2 was administered over 1 h, its toxicity was low regardless of the phase of the cell cycle. Administered separately, 2 Gy irradiation produced only a slight decrease in survival, less marked in the S phase. However, the two agents together induced a synergistic inhibition of the surviving fraction in the S phase when the metal was given immediately after irradiation. If manganese wad administered 3 h after irradiation the two inhibitory effects apparently remained only additive. It seems that MnCl 2 can impair some repair processes starting immediately after irradiation. (orig.)

  5. Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H10; IP25) from Chinese hamster cells

    International Nuclear Information System (INIS)

    D'Anna, J.A.; Gurley, L.R.; Becker, R.R.; Barham, S.S.; Tobey, R.A.; Walters, R.A.

    1980-01-01

    A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approx. 20 amino acids so that the large fragment seen in sodium dodecyl sulfate-acrylamide gels has a molecular weight of approx. 20,000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1 0 , rat H1 0 , and IP 25 , a protein enhanced in differentiated Friend erythroleukemia cells. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP

  6. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation

    Energy Technology Data Exchange (ETDEWEB)

    Holmes, Amie L. [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth Street, P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Joyce, Kellie [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Xie, Hong [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth Street, P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Falank, Carolyne [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); Maine Center for Toxicology and Environmental Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04104-9300, United States of America (United States); and others

    2014-04-15

    Highlights: • The role of homologous recombination repair in DU-induced toxicity was examined. • Loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. • XRCC3 protects cell from DU-induced chromosome breaks and fusions. • XRCC3 plays a role in DU-induced chromosome fragmentation of the X chromosome. - Abstract: Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation.

  7. Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells.

    Science.gov (United States)

    Combs, Rodney G; Yu, Erwin; Roe, Susanna; Piatchek, Michele Bailey; Jones, Heather L; Mott, John; Kennard, Malcolm L; Goosney, Danika L; Monteith, Diane

    2011-01-01

    The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5–3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.

  8. The impact of homologous recombination repair deficiency on depleted uranium clastogenicity in Chinese hamster ovary cells: XRCC3 protects cells from chromosome aberrations, but increases chromosome fragmentation

    International Nuclear Information System (INIS)

    Holmes, Amie L.; Joyce, Kellie; Xie, Hong; Falank, Carolyne

    2014-01-01

    Highlights: • The role of homologous recombination repair in DU-induced toxicity was examined. • Loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. • XRCC3 protects cell from DU-induced chromosome breaks and fusions. • XRCC3 plays a role in DU-induced chromosome fragmentation of the X chromosome. - Abstract: Depleted uranium (DU) is extensively used in both industry and military applications. The potential for civilian and military personnel exposure to DU is rising, but there are limited data on the potential health hazards of DU exposure. Previous laboratory research indicates DU is a potential carcinogen, but epidemiological studies remain inconclusive. DU is genotoxic, inducing DNA double strand breaks, chromosome damage and mutations, but the mechanisms of genotoxicity or repair pathways involved in protecting cells against DU-induced damage remain unknown. The purpose of this study was to investigate the effects of homologous recombination repair deficiency on DU-induced genotoxicity using RAD51D and XRCC3-deficient Chinese hamster ovary (CHO) cell lines. Cells deficient in XRCC3 (irs1SF) exhibited similar cytotoxicity after DU exposure compared to wild-type (AA8) and XRCC3-complemented (1SFwt8) cells, but DU induced more break-type and fusion-type lesions in XRCC3-deficient cells compared to wild-type and XRCC3-complemented cells. Surprisingly, loss of RAD51D did not affect DU-induced cytotoxicity or genotoxicity. DU induced selective X-chromosome fragmentation irrespective of RAD51D status, but loss of XRCC3 nearly eliminated fragmentation observed after DU exposure in wild-type and XRCC3-complemented cells. Thus, XRCC3, but not RAD51D, protects cells from DU-induced breaks and fusions and also plays a role in DU-induced chromosome fragmentation

  9. Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

    Science.gov (United States)

    Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

    2012-01-01

    Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

  10. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    Directory of Open Access Journals (Sweden)

    Han Yong-Mahn

    2009-07-01

    Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

  11. Novel Approach of Differential Staining to Detect Necrotic Cells in Preimplantation Embryos

    Directory of Open Access Journals (Sweden)

    Mohammad Hossein Nasr Esfahani

    2007-01-01

    Full Text Available Background: This novel approach describes a rapid and simple method for identification of necrotic vs. viable cells within a mammalian blastocyst.Materials and Methods: Hatched bovine blastocysts produced in vitro were first incubated for 30 min in pre-equilibrated culture medium containing propidium iodide (PI; 300μg/ml and bisbenzimide (Hoechst: H33342; 5μg/ml fluorescent dyes. Embryos were then freed from residual dyes by thoroughly washing in warm phosphate buffer saline free of calcium and magnesium (PBS-, fixed in 2.5% glutharaldehyde and washed again in PBS- . Stained embryos afterwards were mounted in a drop of glycerol over a microscopic slide. Prepared samples were examined under an epifluorescent microscope using the same excitation wavelength (330-385nm and barrier filter (400nm to distinguish necrosed vs. viable blastomers as being appeared in red and blue, respectively.Results: Obtained results showed that in cells with altered cell membrane such as late apoptotic or necrotic cells, PI and H33342 readily enter through the cytoplasmic barriers and so the chromatin materials are stained by both, but since PI quenches bisbenzimide fluorescence, necrotic blastomeres are seen in red to pinky red, while live cells are seen just as blue.Conclusion: Obtained results clearly indicated that this novel approach can be used as a simple, feasible and precise method for every embryology lab and with all the mammalian blastocysts produced either in vitro or in vivo. The basic assay can be completed in 60 min, and valuable and reliable information can be obtained about the quality of the embryos.

  12. Aryl- and alkyl-phosphorus-containing flame retardants induced mitochondrial impairment and cell death in Chinese hamster ovary (CHO-k1) cells

    International Nuclear Information System (INIS)

    Huang, Chao; Li, Na; Yuan, Shengwu; Ji, Xiaoya; Ma, Mei; Rao, Kaifeng; Wang, Zijian

    2017-01-01

    Phosphorus-containing flame retardants (PFRs) are increasingly in demand worldwide as replacements for brominated flame retardants (BFRs), but insufficient available toxicological information on PFRs makes assessing their health risks challenging. Mitochondria are important targets of various environmental pollutants, and mitochondrial dysfunction may lead to many common diseases. In the present study, mitochondria impairment-related endpoints were measured by a high content screening (HCS) assay for 11 selected non-halogen PFRs in Chinese hamster ovary (CHO-k1) cells. A cluster analysis was used to categorize these PFRs into three groups according to their structural characteristics and results from the HCS assay. Two groups, containing long-chain alkyl-PFRs and all aryl-PFRs, were found to cause mitochondrial impairment but showed different mechanisms of toxicity. Due to the high correlation between cell death and mitochondrial impairment, two PFRs with different structures, trihexyl phosphate (THP) and cresyl diphenyl phosphate (CDP), were selected and compared with chlorpyrifos (CPF) to elucidate their mechanism of inducing cell death. THP (an alkyl-PFR) was found to utilize a similar pathway as CPF to induce apoptosis. However, cell death induced by CDP (an aryl-PFR) was different from classical necrosis based on experiments to discriminate among the different modes of cell death. These results confirm that mitochondria might be important targets for some PFRs and that differently structured PFRs could function via distinct mechanisms of toxicity. - Highlights: • Mitochondrial impairment induced by PFRs was observed in CHO-k1 cells. • THP (an alkyl-PFR) induced a caspase-mediated apoptosis in CHO-k1 cells. • The cell death induced by CDP (an aryl-PFR) was not traditional apoptosis or necrosis.

  13. No specific gene expression signature in human granulosa and cumulus cells for prediction of oocyte fertilisation and embryo implantation.

    Directory of Open Access Journals (Sweden)

    Tanja Burnik Papler

    Full Text Available In human IVF procedures objective and reliable biomarkers of oocyte and embryo quality are needed in order to increase the use of single embryo transfer (SET and thus prevent multiple pregnancies. During folliculogenesis there is an intense bi-directional communication between oocyte and follicular cells. For this reason gene expression profile of follicular cells could be an important indicator and biomarker of oocyte and embryo quality. The objective of this study was to identify gene expression signature(s in human granulosa (GC and cumulus (CC cells predictive of successful embryo implantation and oocyte fertilization. Forty-one patients were included in the study and individual GC and CC samples were collected; oocytes were cultivated separately, allowing a correlation with IVF outcome and elective SET was performed. Gene expression analysis was performed using microarrays, followed by a quantitative real-time PCR validation. After statistical analysis of microarray data, there were no significantly differentially expressed genes (FDR<0,05 between non-fertilized and fertilized oocytes and non-implanted and implanted embryos in either of the cell type. Furthermore, the results of quantitative real-time PCR were in consent with microarray data as there were no significant differences in gene expression of genes selected for validation. In conclusion, we did not find biomarkers for prediction of oocyte fertilization and embryo implantation in IVF procedures in the present study.

  14. Role of repair saturation in the response of plateau-phase Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Braby, L.A.; Nelson, J.M.; Metting, N.F.

    1987-01-01

    Two repair rates are seen in split-dose experiments on starved plateau-phase CHO cells. It has been assumed that this indicates two different processes repairing two distinct types of sublethal damage. However results of experiments at different dose levels are not consistent with models that assume that the damage is entirely sublethal. Another hypothesis that has been considered is the saturation of a repair mechanism having a limited pool of repair enzymes. Such saturation phenomena have been observed in biochemical repair studies and have thus formed the basis for a model of cellular response, which was shown to be capable of producing dose response curves in good agreement with experimental observations. This model can be extended to account for both dose-rate and split-dose effects

  15. Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

    Directory of Open Access Journals (Sweden)

    Ryutaro Hirasawa

    Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

  16. Response to high LET radiation 12C (LET, 295 keV/microm) in M5 cells, a radio resistant cell strain derived from Chinese hamster V79 cells.

    Science.gov (United States)

    Pathak, R; Sarma, A; Sengupta, B; Dey, S K; Khuda-Bukhsh, A R

    2007-01-01

    To study the effects of 12C-beam of 295 keV/microm (57.24 MeV) on M5 and Chinese hamster V79 cells by using cytogenetic assays like micronuclei (MN) induction, chromosomal aberrations (CA) and apoptosis. Additionally, the relative survival of these two cell lines was tested by the colony forming ability of the cells, with a view to understanding the mechanism of cellular damages that lead to difference in cell survival. Confluent cells were irradiated with 12C-beam at various doses using 15UD Pelletron accelerator. Cell survival was studied by the colony forming ability of cells. MN assay was done by fluorescent staining. Different types of chromosomal aberrations in metaphase cells were scored at 12 h after irradiation. Apoptosis was measured at different post irradiation times as detected by nuclear fragmentation and DNA ladder was prepared after 48 h of incubation. Dose-dependent decrease in surviving fractions was found in both the cell lines. However, the surviving fractions were higher in M5 cells in comparison to V79 cells when exposed to the same radiation doses. On the other hand, induced MN frequencies, CA frequencies and apoptosis percentages were less in M5 cells than V79 cells. Very good correlations between surviving fractions and induced MN frequencies or induced total CA or induced apoptosis percentages were obtained in this study. The cell strain M5 showed relatively more radio-resistance to 12C-beam compared to Chinese hamster V79 cells in this study. As the MN formation, CA and apoptosis induction were less in M5 cells as compared to parental V79 cells, the higher cell survival in the former could possibly be attributed to their better repairing ability leading to higher cell survival.

  17. Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Peura, T.T.; Hartwich, K.M.

    2005-01-01

    The processes of cellular differentiation were studied in somatic cell nuvlear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos...... were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients...

  18. Enhancement of postreplication repair in ultraviolet-light-irradiated Chinese hamster cells by irradiation in G2 or s-phase

    International Nuclear Information System (INIS)

    D'Ambrosio, S.M.; Aebersold, P.M.; Setlow, R.B.

    1978-01-01

    Postreplication repair in synchronous Chinese hamster cells was determined after split doses of ultraviolet (uv) radiation. Repair was enhanced by irradiation of cells in G 2 or S-phase with a small dose of uv radiation at least 1.5 h before a three-fold larger dose of uv. There was significantly greater enhancement when the first dose was given in G 2 than when it was given in the S-phase 0.5 to 1.5 h before the test dose. These data indicate that enhancement of postreplication repair does not require active DNA replication and qualitatively is independent of when in the cell cycle the cells are irradiated

  19. Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent, monobromobimane

    International Nuclear Information System (INIS)

    Fleming, A.D.; Cummins, J.M.; Kuehl, T.J.; Seidel, G.E. Jr.; Yanagimachi, R.

    1986-01-01

    Cauda epididymal spermatozoa of the golden hamster were labelled with the thiol-alkylating reagent, monobromobimane (MB). Female hamsters underwent uterine insemination with labelled spermatozoa at laparotomy under metofane anesthesia. All 12 females examined between 5 and 54 h postinsemination yielded a total of 83/100 (83%) eggs in the process of fertilization or embryos. Under ultraviolet (UV) exposure all exhibited a fluorescent tail which, in the 4- and 8-cell embryos, could be seen to be fraying or disintegrating. As cleavage progressed, labelled tail components came to be restricted among the blastomeres such that at the 4- and 8-cell stage the tail could be seen in only one to three blastomeres. To study complete development and pregnancy another 12 females received uterine insemination. After recovery from anesthesia (approximately equal to 4 h) these females were mated with a vasectomized male bearing a dominant genetic marker (black eyes) to allow unequivocal determination of paternity in the fetuses and young produced. Seven became pregnant with one female losing her pregnancy about Day 7 of gestation. Two females sacrificed on Day 13 produced 17 normal fetuses and one resorption. Four females delivered 16 young, all normal at birth and in subsequent growth and fertility. In addition, insemination of female rabbits with MB-labelled spermatozoa yielded normal embryos (50/52 96%) from 3 does on Day 2 and (38/64 60%) from 4 does on Days 4 or 5. Two normal litters (9 bunnies) have delivered from 3 does allowed to carry to term

  20. Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

    2009-01-01

    Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

  1. Experimental tumor growth of canine osteosarcoma cell line on chick embryo chorioallantoic membrane (in vivo studies).

    Science.gov (United States)

    Walewska, Magdalena; Dolka, Izabella; Małek, Anna; Wojtalewicz, Anna; Wojtkowska, Agata; Żbikowski, Artur; Lechowski, Roman; Zabielska-Koczywąs, Katarzyna

    2017-05-12

    The chick embryo chorioallantoic membrane (CAM) model is extensively used in human medicine in preclinical oncological studies. The CAM model has several advantages: low cost, simple experimental approach, time saving and following "3R principles". Research has shown that the human osteosarcoma cell lines U2OS, MMNG-HOS, and SAOS can form tumors on the CAM. In veterinary medicine, this has been described only for feline fibrosarcomas, feline mammary carcinomas and canine osteosarcomas. However, in case of canine osteosarcomas, it has been shown that only non-adherent osteosarcoma stem cells isolated from KTOSA5 and CSKOS cell lines have the ability to form microtumors on the CAM after an incubation period of 5 days, in contrast to adherent KTOSA5 and CSKOS cells. In the presented study, we have proven that the commercial adherent canine osteosarcoma cell line (D-17) can form vascularized tumors on the CAM after the incubation period of 10 days.

  2. Acceptors for poly(ADP-ribose) in irradiated Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Xue, L.Y.; Sokany, N.M.; Friedman, L.R.; Oleinick, N.L.

    1985-01-01

    Strand breaks in DNA, as produced by ionizing radiation, stimulate the synthesis of poly(ADP-ribose) (pADPR) by the nuclear enzyme pADPR transferase (ADPRT). The polymer is covalently bound to chromatin-associated proteins and may function in repair of DNA lesions. When total /sup 32/P-pADPR-protein is analyzed by electrophoresis on SDS-polyacrylamide gels, the major radioactive bands correspond to the 116 kD ADPRT and the low molecular weight (histone) region. On two-dimensional gels (isoelectric focusing followed by SDS-PAGE) several ADP-ribosylated species can be detected in each molecular weight range. The intensity of label in each species is greater for proteins isolated from irradiated (10 or 100 Cy) rather than control cells. For detailed analysis of histones, the authors incubated isolated nuclei with /sup 32/P-NAD, extracted histones in acid, and subjected them to electrophoresis in acid-urea gels. Specific radiation-induced increases in pADPR were seen on some nucleosomal core histone bands but not on histone H1. The results suggest that radiation-induced strand breaks stimulate ADPRT to modify core histones; the resultant increase in negative charge could loosen nucleosomal structure, permitting access of repair enzymes to the DNA lesions

  3. Regional assignment of seven genes on chromosome 1 of man by use of man-Chinese hamster somatic cell hybrids. I. Results obtained after hybridization of human cells carrying reciprocal translocations involving chromosome 1.

    Science.gov (United States)

    Jongsma, A P; Burgerhout, W G

    1977-01-01

    Regional localization studies of genes coding for human PGD, PPH1, PGM1, UGPP, GuK1, Pep-C, and FH, which have been assigned to chromosome 1, were performed with man-Chinese hamster somatic cell hybrids, Informative hybrids that retained fragments of the human chromosome 1 were produced by fusion of hamster cells with human cells carrying reciprocal translocations involving chromosome 1. Analysis of the hybrids that retained one of the translocation chromosomes or de novo rearrangements involving the human 1 revealed the following gene positions: PGD and PPH1 in 1pter leads to 1p32, PGM1 in 1p32 leads to 1p22, UGPP and GuK1 in 1q21 leads to 1q42, FH in 1qter leads to 1q42, and Pep-C probably in 1q42.

  4. Co-culture of human embryos with autologous cumulus cell clusters and its beneficial impact of secreted growth factors on preimplantation development as compared to standard embryo culture in assisted reproductive technologies (ART

    Directory of Open Access Journals (Sweden)

    Alexandros Vithoulkas

    2017-12-01

    Conclusion(s: The investigated factors, among other substances, may be causally connected to the beneficial effect observed on embryo development. Our findings suggest that co-culture with autologous cumulus cell clusters improves the outcome of embryo culture in IVF programs.

  5. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  6. Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Wellner, R.B.; Ghosh, P.C.; Roecklein, B.; Wu, H.C.

    1987-01-01

    Increased [ 3 H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary and yeast mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [ 3 H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [ 3 H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [ 3 H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [ 3 H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [ 3 H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [ 3 H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [ 3 H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [ 3 H]palmitate incorporation into the 20,000 molecular weight species

  7. Perkembangan Praimplantasi Embrio Mencit dengan Materi Genetik yang Berasal dari Parental, Maternal, dan Inti Sel Somatik (PRE-IMPLANTATION DEVELOPMENT OF MOUSE EMBRYO WITH GENETIC MATERIAL DERIVED FROM PARENTAL, MATERNAL AND SOMATIC CELL NUCLEUS

    Directory of Open Access Journals (Sweden)

    Harry Murti

    2014-05-01

    Full Text Available Cloned embryo and parthenogenetic embryo are a potential source of stem cells for regenerativemedicine. Stem cells derived from those embryos are expected to overcome the ethical issues to the use offertilization embryos for therapeutic purposes. The pre-implantation development is a critical step fordeveloping embryos reach the blastocyst stage. The objectives in vivo of this research are to produce mousecloned embryo, parthenogenetic embryo, and fertilized embryo and to study stages of  in vitro pre-implantation development culture. In vivo fertilized embryos, mouse oocytes, and cumulus cells were usedin this study. Treatment was performed on female mice superovulated with PMSG and hCG injections.Two-cell stage of in vivo fertilized embryos were collected on the second day post hCG injection. Clonedembryos were produced through Somatic Cell Nuclear Transfer (SCNT, which included enucleation, nucleartransfer and artificial activation. Parthenogenetic embryos were produced with artificial activationtechnique. The result of the research indicated that SCNT application was able to produce cloned embryos which could develop to blastocyst stage (3,2%. In addition, artificial activation of oocytes could produceparthenogenetic embryos which were able to develop up to the blastocyst stage (8,6%. In conclusion,efficiency level of parthenogenetic embryos that is able to reach the blastocyst stage was higher than in thecloned embryos. Fertilized embryos shows a better development and more efficient compared to in vitrocloned embryos and parthenogenetic embryos cultures.

  8. Proteomic analysis of the early bovine yolk sac fluid and cells from the day 13 ovoid and elongated preimplatation embryos

    DEFF Research Database (Denmark)

    Jensen, Pernille L.; Beck, Hans Christian; Petersen, Tonny S.

    2014-01-01

    differentiate into the hypoblast and epiblast, which remain surrounded by the trophectoderm. The formation of the hypoblast epithelium is also accompanied by a change in the fluid within the embryo, i.e., the blastocoel fluid gradually alters to become the primitive yolk sac (YS) fluid. Our previous research......The bovine blastocyst hatches 8 to 9 days after fertilization, and this is followed by several days of preimplantation development during which the embryo transforms from a spherical over an ovoid to an elongated shape. As the spherical embryo enlarges, the cells of the inner cell mass...... describes the protein composition of human and bovine blastocoel fluid, which is surrounded by the trophectoderm and undifferentiated cells of the inner cell mass. In this study, we further examine the changes in the protein composition in both the primitive YS fluid and the embryonic cells during early...

  9. Ethanol exposure affects cell movement during gastrulation and induces split axes in zebrafish embryos.

    Science.gov (United States)

    Zhang, Ying; Shao, Ming; Wang, Lifeng; Liu, Zhongzhen; Gao, Ming; Liu, Chao; Zhang, Hongwei

    2010-06-01

    To explore the toxic effects of ethanol on axis formation during embryogenesis, zebrafish embryos at different developmental stages were treated with 3% ethanol for 3h. The effects of ethanol exposure appeared to be stage-dependent. The dome stage embryo was most sensible to form posterior split axes upon ethanol exposure. Morphological and histological observations and whole-mount in situ hybridization results showed that ethanol exposure at this stage caused a general gastrulation delay, and induced double notochords, double neural tubes and two sets of somites in the posterior trunk. Mechanistically, no ectopic organizer was found by examining the expression patterns of dorsoventral markers including goosecoid, chordin and eve1 at the onset of gastrulation. However, radial intercalation, epiboly and convergence extension were inhibited by ethanol exposure as revealed by cell labeling, phenotypic observation and the expression patterns of axial or paraxial markers. Further investigation showed that the cell aggregation might be affected by ethanol exposure, as indicated by the much more scattered expression pattern of chordin, eve1 and wnt11 at the early gastrula stage, and the discontinuous gsc positive cells during migration. These results imply that ethanol might affect cell movement before and during gastrulation and as a consequence, induces a split axes phenotype. Copyright 2010 ISDN. Published by Elsevier Ltd. All rights reserved.

  10. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    Directory of Open Access Journals (Sweden)

    Binghua Xue

    Full Text Available Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

  11. Transfection of Chinese hamster ovary DHFR/sup -/ cells with the gene coding for heat shock protein 70 from drosophila melanogaster

    International Nuclear Information System (INIS)

    Duffy, J.J.; Carper, S.W.; Gerner, E.W.

    1987-01-01

    Chinese hamster ovary DHFR/sup -/ cells (CHO-DHFR/sup -/) were transfected with the plasmid pSV2-dhfr expressing the mouse gene coding for dhfr or with the same plasmid containing the gene coding for the Drosophila melanogaster heat shock protein 70 (hsp70), pSVd-hsp70. Three subcloned cell lines selected for expression of the dhfr gene were shown to contain either the vector sequence (G cells) or varying copies of pSVd-hsp70 (H cells). One line of H cells was shown to contain > 30 copies of the D. melanogaster hsp70 gene and to express the hsp70 RNA at significant levels. No difference between G and H cells was observed in the rate of growth, in the development of thermotolerance, or in the sensitivity of actin microfilament bundles to heat shock. However, H cells containing the transfected hsp70 gene had an altered morphology when compared to the G cells and the parental CHO-DHFR/sup -/ cells being more fibroblastic. The adhesion properties of the H cells was also decreased when compared to the G cells. These results show that insertion of the D. melanogaster gene into CHO cells does not effect growth rates or heat shock responses but may alter cell morphology and adhesion

  12. Developmental block and programmed cell death in Bos indicus embryos: effects of protein supplementation source and developmental kinetics.

    Directory of Open Access Journals (Sweden)

    Sheila Merlo Garcia

    Full Text Available The aims of this study were to determine if the protein source of the medium influences zebu embryo development and if developmental kinetics, developmental block and programmed cell death are related. The culture medium was supplemented with either fetal calf serum or bovine serum albumin. The embryos were classified as Fast (n = 1,235 or Slow (n = 485 based on the time required to reach the fourth cell cycle (48 h and 90 h post insemination - hpi -, respectively. The Slow group was further separated into two groups: those presenting exactly 4 cells at 48 hpi (Slow/4 cells and those that reached the fourth cell cycle at 90 hpi (Slow. Blastocyst quality, DNA fragmentation, mitochondrial membrane potential and signs of apoptosis or necrosis were evaluated. The Slow group had higher incidence of developmental block than the Fast group. The embryos supplemented with fetal calf serum had lower quality. DNA fragmentation and mitochondrial membrane potential were absent in embryos at 48 hpi but present at 90 hpi. Early signs of apoptosis were more frequent in the Slow and Slow/4 cell groups than in the Fast group. We concluded that fetal calf serum reduces blastocyst development and quality, but the mechanism appears to be independent of DNA fragmentation. The apoptotic cells detected at 48 hpi reveal a possible mechanism of programmed cell death activation prior to genome activation. The apoptotic cells observed in the slow-developing embryos suggested a relationship between programmed cell death and embryonic developmental kinetics in zebu in vitro-produced embryos.

  13. Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line.

    Science.gov (United States)

    Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

    2010-06-01

    Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H(2)O(2)-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H(2)O(2)-induced cell damage in vitro.

  14. The forces that shape embryos: physical aspects of convergent extension by cell intercalation

    International Nuclear Information System (INIS)

    Keller, Ray; Shook, David; Skoglund, Paul

    2008-01-01

    We discuss the physical aspects of the morphogenic process of convergence (narrowing) and extension (lengthening) of tissues by cell intercalation. These movements, often referred to as 'convergent extension', occur in both epithelial and mesenchymal tissues during embryogenesis and organogenesis of invertebrates and vertebrates, and they play large roles in shaping the body plan during development. Our focus is on the presumptive mesodermal and neural tissues of the Xenopus (frog) embryo, tissues for which some physical measurements have been made. We discuss the physical aspects of how polarized cell motility, oriented along future tissue axes, generate the forces that drive oriented cell intercalation and how this intercalation results in convergence and extension or convergence and thickening of the tissue. Our goal is to identify aspects of these morphogenic movements for further biophysical, molecular and cell biological, and modeling studies

  15. Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus

    DEFF Research Database (Denmark)

    Li, Yiping; Handberg, K.J.; Juul-Madsen, H.R.

    2007-01-01

    Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease in chickens and causes a significant economic loss for the poultry industry. Little is understood about the mechanism involved in the host responses to IBDV infection. For better understanding the IBDV......-host interaction, we measured steady-state levels of transcripts from 28 cellular genes of chicken embryo (CE) cell cultures infected with IBDV vaccine stain Bursine-2 during a 7-day infection course by use of the quantitative real-time RT-PCR SYBR green method. Of the genes tested, 21 genes (IRF-1, IFN 1...

  16. Chromosomal mutations and chromosome loss measured in a new human-hamster hybrid cell line, ALC: studies with colcemid, ultraviolet irradiation, and 137Cs gamma-rays

    Science.gov (United States)

    Kraemer, S. M.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

    1997-01-01

    Small mutations, megabase deletions, and aneuploidy are involved in carcinogenesis and genetic defects, so it is important to be able to quantify these mutations and understand mechanisms of their creation. We have previously quantified a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in a hamster-human hybrid cell line AL. S1- mutants have lost expression of a human cell surface antigen, S1, which is encoded by the M1C1 gene at 11p13 so that mutants can be detected via a complement-mediated cytotoxicity assay in which S1+ cells are killed and S1- cells survive. But loss of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the AL hybrid, so that mutants that have lost the entire chromosome 11 die and escape detection. To circumvent this, we fused AL with Chinese hamster ovary (CHO) cells to produce a new hybrid, ALC, in which the requirement for maintaining 11p15.5 is relieved, allowing us to detect mutations events involving loss of 11p15.5. We evaluated the usefulness of this hybrid by conducting mutagenesis studies with colcemid, 137Cs gamma-radiation and UV 254 nm light. Colcemid induced 1000 more S1- mutants per unit dose in ALC than in AL; the increase for UV 254 nm light was only two-fold; and the increase for 137Cs gamma-rays was 12-fold. The increase in S1- mutant fraction in ALC cells treated with colcemid and 137Cs gamma-rays were largely due to chromosome loss and 11p deletions often containing a breakpoint within the centromeric region.

  17. Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

    Science.gov (United States)

    Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

    2014-08-01

    Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, pvitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, pVitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

  18. Radiobiological properties of radiosensitive XR-1 Chinese hamster cells and hybrids from these and human A-T cells

    International Nuclear Information System (INIS)

    Bahari, I.B.

    1989-01-01

    Results indicate that XR-1 cells were very radiosensitive to gamma-irradiation compared to its parental type, and that this radiosensitivity is cell cycle dependent. Irradiating the cells the G 1 or plateau phase did not induce any delay entering S-phase but mitotic delays were observed in both XR-1 and the wild-type cells. The delays per unit dose were much longer for XR-1. A delay in subculture from plateau phase reduced the mitotic delay in both cell lines. Unlike the wild-type cells which expressed virtually all chromosome-type aberrations after irradiation of G 1 cells, the XR-1 cells expressed both chromatid- as well as chromosome-type aberrations. There was a one-to-one correlation between total aberrations induced and lethality for both cells. Many of these radiobiological properties of XR-1 cells relative to the wild-type cells, mimic the response of A-T cells relative to the normal human cells. However, the restoration of radioresistance and cytogenetic response in the XR1/AT5BI(4) hybrid cells suggest that the XR-1 and A-T cells have different defects because of the complementation in the hybrids. It also appears that this genetic defect is recessive in nature

  19. Use of the α-mannosidase I inhibitor kifunensine allows the crystallization of apo CTLA-4 homodimer produced in long-term cultures of Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Yu, Chao; Crispin, Max; Sonnen, Andreas F.-P.; Harvey, David J.; Chang, Veronica T.; Evans, Edward J.; Scanlan, Christopher N.; Stuart, David I.; Gilbert, Robert J. C.; Davis, Simon J.

    2011-01-01

    The α-mannosidase I inhibitor kifunensine inhibited N-glycan processing in long-term cultures of Chinese hamster ovary cells, allowing deglycosylation and crystallization of the homodimeric extracellular region of the inhibitory glycoprotein receptor CTLA-4 (CD152). Glycoproteins present problems for structural analysis since they often have to be glycosylated in order to fold correctly and because their chemical and conformational heterogeneity generally inhibits crystallization. It is shown that the α-mannosidase I inhibitor kifunensine, which has previously been used for the purpose of glycoprotein crystallization in short-term (3–5 d) cultures, is apparently stable enough to be used to produce highly endoglycosidase H-sensitive glycoprotein in long-term (3–4 week) cultures of stably transfected Chinese hamster ovary (CHO) cells. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based analysis of the extracellular region of the cytotoxic T-lymphocyte antigen 4 (CTLA-4; CD152) homodimer expressed in long-term CHO cell cultures in the presence of kifunensine revealed that the inhibitor restricted CTLA-4 glycan processing to Man 9 GlcNAc 2 and Man 5 GlcNAc 2 structures. Complex-type glycans were undetectable, suggesting that the inhibitor was active for the entire duration of the cultures. Endoglycosidase treatment of the homodimer yielded protein that readily formed orthorhombic crystals with unit-cell parameters a = 43.9, b = 51.5, c = 102.9 Å and space group P2 1 2 1 2 1 that diffracted to Bragg spacings of 1.8 Å. The results indicate that kifunensine will be effective in most, if not all, transient and long-term mammalian cell-based expression systems

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  1. X-ray induction of 6-thioguanine-resistant mutants in division arrested, G0/G1 phase Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    O' Neill, J.P.; Flint, K.B.

    The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G0/G1 phase of the cell cycle through 9 days incubation in serum-free medium. In comparison with exponential phase cultures, the arrested cells showed increased cytotoxicity and mutation induction over the dose range of 50-800 rad. Exponential cultures showed a linear mutant frequency-survival relationship while the arrested cells showed a biphasic linear relationship. A post irradiation holding period 24 h does not result in any change in the mutant frequency. The increased sensitivity of the arrested cells to the mutagenic effects of X-rays appears to be a cell-cycle phase phenomenon. Upon readdition of serum, the arrested cells re-enter the cell cycle in a synchronous manner, reaching S phase at 10-12 h. Cells irradiated at 5 h after serum addition, i.e. in G1, show a similar dose response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G1, S or G2, show lower mutation induction. These observations are consistent with a chromosome interchange mechanism of mutation induction by X-rays, possibly through interactions between repairing regions of the DNA. Irradiation of cells in the G0/G1 phase allow more time for such interactions in the absence of semiconservative DNA replication. (orig.).

  2. Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos.

    Science.gov (United States)

    Akshey, Y S; Malakar, D; De, A Kumar; Jena, M Kumar; Pawar, S Kumar; Dutta, R; Sahu, S

    2011-05-01

    The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups-contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods-one half by 2 μM Ca ionophore and another half by 2.31 kV/cm for 15 μSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Human chromosome-specific changes in a human-hamster hybrid cell line (AL) assessed by fluorescent in situ hybridization (fish)

    International Nuclear Information System (INIS)

    Geard, Charles R.; Jenkins, Gloria

    1995-01-01

    Purpose: To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. Methods and Materials: Synchronized human-hamster hybrid cells containing human chromosome 11 were obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%) were irradiated with 137 Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells collected 16-20 h later; chromosomal spreads were prepared, denatured, and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. Results: Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatible with mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosome per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosome was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosome was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40-60%) incidence of incomplete exchanges between human and hamster chromosomes. Conclusions: Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1

  4. Induction and removal of DNA interstrand cross-links in V-79 Chinese hamster cells measured by hydroxylapatite chromatography after treatments with bifunctional furocoumarins

    International Nuclear Information System (INIS)

    Dardalhon, M.; Averbeck, D.

    1988-01-01

    DNA interstrand crosslinks (CL) photoinduced by bifunctional furocoumarins in V-79 Chinese hamster cells were measured by alkaline denaturation and hydroxylapatite chromatography. Treatments with 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (4,5',8-TMP) and 365 nm irradiation (UVA) confer a dose-dependent linear increase in the amount of double-stranded DNA indicating the induction of CL. Determination in alkaline sucrose gradients of the molecular weight of the DNA and estimation of drug-induced strand breakage allowed quantification of the CL induced. 5-MOP was found to be slightly more effective than 8-MOP whereas 4,5',8-TMP was 9 times more effective for the induction of CL. The fate of CL during post-treatment incubation was also followed. Cells in exponential growth phase were found to be efficient in the removal of CL. (Author)

  5. X-radiation-induced transformation in a C3H mouse embryo-derived cell line

    International Nuclear Information System (INIS)

    Terzaghi, M.; Little, J.B.

    1976-01-01

    Reproducible x-ray-induced oncogenic transformation has been demonstrated in an established cell line of mouse embryo fibroblasts. Cells derived from transformed foci formed malignant tumors when injected into syngeneic hosts. An exponential increase in the number of transformants per viable cell occurred with doses of up to 400 rads of x-radiation. The transformation frequency in exponentially growing cultures remained constant at 2.3 x 10 -3 following doses of 400 to 1500 rads. There was little change in survival following x-ray doses up to 300 rads. Doses greater than 300 rads were associated with an exponential decline in survival; the D 0 for the survival curve was 175 rads. Transformation frequency varied with changes in the number of viable cells seeded per dish. There was about a 10-fold decline in the transformation frequency when the number of cells was increased from 400 to 1000 viable cells/100-mm Petri dish. Below this density range there was little change in transformation frequency. The presence of lethally preirradiated cells was not associated with an enhancement of transformation in irradiated cells or with the induction of transformation in unirradiated cell cultures. Amphotericin B (Fungizone) inhibited the appearance of transformants when added to the culture medium within 2 to 3 weeks after initiation of the experiment

  6. Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

    DEFF Research Database (Denmark)

    Jeong, J; Han, I; Lim, Y

    2001-01-01

    of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C...

  7. Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

    Directory of Open Access Journals (Sweden)

    Lin Yuan

    2014-11-01

    Full Text Available Cloned pigs generated by somatic cell nuclear transfer (SCNT show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP. q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.

  8. Aberrant Expression of Xist in Aborted Porcine Fetuses Derived from Somatic Cell Nuclear Transfer Embryos

    Science.gov (United States)

    Yuan, Lin; Wang, Anfeng; Yao, Chaogang; Huang, Yongye; Duan, Feifei; Lv, Qinyan; Wang, Dongxu; Ouyang, Hongsheng; Li, Zhanjun; Lai, Liangxue

    2014-01-01

    Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos. PMID:25429426

  9. Envisaging the embryo in stem cell research: rhetorical strategies and media reporting of the ethical debates.

    Science.gov (United States)

    Williams, Clare; Kitzinger, Jenny; Henderson, Lesley

    2003-11-01

    How is the embryo defined, envisaged, imagined? Who speaks on its behalf, and how? Based on a study of UK press and TV news reporting, this paper identifies the rhetorical strategies used to assert competing ethical positions around embryonic stem cell research. We show how both sides in the dispute mobilise metaphors and use personification to recruit support; and how they promote different ideas about the embryo's significance, size, and social embeddedness and present competing narratives about its origins, destiny and 'death'. The role of visual representation is key here. It does not follow the usual pattern whereby, in the abortion debate, those 'on the side' of the fetus display its image while those who are 'pro-choice' shy away from this. In the stem cell debate the pattern is inverted, highlighting the role of technologies of visualisation in defining what counts as human. Our analysis also shows how the media coverage marginalises women's perspectives, disregards more fundamental challenges to science, side-lines concerns about effectiveness or safety and curtails discussion of broader issues. We reflect on the media processes restricting debate in this way and conclude by identifying opportunities for a more inclusive discussion of science ethics.

  10. Delayed cell death, giant cell formation and chromosome instability induced by X-irradiation in human embryo cells

    International Nuclear Information System (INIS)

    Roy, K.; Kodama, Seiji; Suzuki, Keiji; Watanabe, Masami

    1999-01-01

    We studied X-ray-induced delayed cell death, delayed giant cell formation and delayed chromosome aberrations in normal human embryo cells to explore the relationship between initial radiation damage and delayed effect appeared at 14 to 55 population doubling numbers (PDNs) after X-irradiation. The delayed effect was induced in the progeny of X-ray survivors in a dose-dependent manner and recovered with increasing PDNs after X-irradiation. Delayed plating for 24 h post-irradiation reduced both acute and delayed lethal damage, suggesting that potentially lethal damage repair (PLDR) can be effective for relieving the delayed cell death. The chromosome analysis revealed that most of the dicentrics (more than 90%) observed in the progeny of X-ray survivors were not accompanied with fragments, in contrast with those observed in the first mitosis after X-irradiation. The present results indicate that the potentiality of genetic instability is determined during the repair process of initial radiation damage and suggest that the mechanism for formation of delayed chromosome aberrations by radiation might be different from that of direct radiation-induced chromosome aberrations. (author)

  11. Assignment of genes to chromosome 4 of the River buffalo with a panel of buffalo-hamster hybrid cells.

    Science.gov (United States)

    Nahas, S M; Hondt, H A; Othman, O S; Bosma, A A; Haan, N A

    1993-01-12

    To identify the river buffalo chromosome carrying the genes coding for GAPD, TPI1, and LDHB, karyotypic examination was carried out on 14 buffalo-hamster hybrid clones previously tested for presence of this syntenic group. In cattle, this group (U3) has been assigned to chromosome 5, which is assumed to be homologous to the long arm of buffalo chromosome 4. Chromosome 4 was present in all five clones expressing the three enzymes, and absent in all seven negative clones, indicating that in the buffalo GAPD, TPI1, and LDHB are located on chromosome 4. One clone, expressing GAPD and TPI1, but not LDHB, was found to carry a translocation between hamster marker chromosome M(2) and buffalo 4q1 → 4qter. In another clone, expressing LDHB, but not GAPD and TPI1, chromosome 4 was absent, while a very small, unidentifiable acrocentric was present. These observations suggest that LDHB is located in the proximal part of 4q1, and that GAPD and TPI1 are located more distally, in 4q1 → 4q2. ZUSAMMENFASSUNG: Lokalisierung von Genen auf Chromosom 4 des Flußbüffels durch Büffel-Hamster-Hybridzellen Zur Identifikation von Flußbüffelchromosomen mit Genen für GAPD, TPI1 und LDHB wurden Karyotypenbestimmungen an 14 Büffel-Hamster-Hybridklonen durchgeführt, die vorher auf Anwesenheit der betreffenden synthenischen Gruppen geprüft worden waren. Bei Rindern wird diese Gruppe (U3) dem Chromosom 5 zugeordnet, welches als homolog mit dem langen Arm des Büffelchromosoms 4 betrachtet wird. Chromosom 4 war in allen fünf Klonen, die die drei Enzyme exprimiert haben, vorhanden und fehlte in allen sieben negativen klonen, so daß angenommen werden kann, daß sich bei Büffeln GAPD, TPI1 und LDHB auf Chromosom 4 befinden. Bei einem Klon, der GAPD und TPI1, aber nicht LDHB zeigte, wurde eine Translokation zwischen dem Hamstermarkerchromosom M2 und Büffel 4q1 → 4qter gefunden. Im einem anderen Klon, der LDHB, nicht aber GAPD und TPI1 zeigte, war Chromosom 4 nicht vorhanden, wohl aber

  12. A uv-sensitive Chinese hamster lung fibroblast cell line (V79/UC) with a possible defect in DNA polymerase activity is deficient in DNA repair

    International Nuclear Information System (INIS)

    Creissen, D.M.; Hill, C.K.

    1991-01-01

    Studies of repair enzyme activities in a uv-sensitive cell line (V79/UC) derived from Chinese hamster V79 cells have revealed levels of total DNA polymerase that are about 50% of the levels in the parental cell line. There are a number of DNA polymerase inhibitors available which allow us to distinguish between the major forms of DNA polymerase (alpha, beta, gamma, and delta) identified in mammalian cells. Enzyme assays with these inhibitors indicate that the aphidicolin-sensitive DNA polymerase is defective in the V79/UC cell line. This could be either polymerase alpha or delta, or both. The V79/UC cells do not express resistance to aphidicolin in standard toxicity studies. However, when aphidicolin is added postirradiation in survival assays designed to measure the extent of inhibitable repair, V79/UC cells do not respond with the further decrease in survival seen in the parental line. Further evidence of a polymerase-dependent repair defect is evident from alkaline elution data. In this case the V79/UC cells show the appearance of single-strand breaks following uv irradiation in the absence of any added inhibitor. Cells of the V79/M12G parental line, on the other hand, show the appearance of single-strand breaks only when aphidicolin is present

  13. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

    Science.gov (United States)

    Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

    2016-08-25

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

  14. Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo

    Directory of Open Access Journals (Sweden)

    McCarthy John J

    2005-08-01

    Full Text Available Abstract Background Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. Results We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. Conclusions Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.

  15. Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

    Science.gov (United States)

    Sugimura, S; Narita, K; Yamashiro, H; Sugawara, A; Shoji, T; Terashita, Y; Nishimori, K; Konno, T; Yoshida, M; Sato, E

    2009-09-01

    Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (Ptip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

  16. Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

    Science.gov (United States)

    Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

    2013-02-01

    Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

  17. Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex

    DEFF Research Database (Denmark)

    Holm, Peter; Shukri, Naseer Mahmoud; Vajta, Gabor

    1998-01-01

    The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g...

  18. Development, DNA fragmentation and cell death in porcine embryos afer 24 h storage under different conditions

    NARCIS (Netherlands)

    Rubio Pomar, F.J.; Ducro-Steverink, D.W.B.; Hazeleger, W.; Teerds, K.J.; Colenbrander, B.; Bevers, M.M.

    2004-01-01

    For practical applications of porcine embryo transfer (ET) it is important to develop feasible embryo storage conditions. The aim of the present study was to evaluate the effect of short-term storage (24 h) on the quality of in vivo produced porcine embryos. Three temperatures 18, 25 and 38 degreesC

  19. Effect of Bcl-xL overexpression on sialylation of Fc-fusion protein in recombinant Chinese hamster ovary cell cultures.

    Science.gov (United States)

    Lee, Jong Hyun; Kim, Yeon-Gu; Lee, Gyun Min

    2015-01-01

    The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl-xL overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc-fusion protein, which were derived from DUKX-B11 and DG44, respectively, were engineered to have regulated Bcl-xL overexpression using the Tet-off system. For both cell lines, Bcl-xL overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc-fusion protein titer increased by Bcl-xL overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl-xL overexpression, the sialylation of Fc-fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl-xL overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl-xL overexpression in rCHO cell culture increased Fc-fusion protein production and also reduced the impairment of sialylation of Fc-fusion protein by maintaining high viability during batch cultures. © 2015 American Institute of Chemical Engineers.

  20. The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XR-V15B

    International Nuclear Information System (INIS)

    Helbig, R.; Speit, G.; Zdzienicka, M.Z.

    1995-01-01

    The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible change in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS. 28 refs., 4 figs., 3 tabs

  1. Survival and mutation in clones derived from V79 Chinese hamster cells irradiated with multiple small exposures to far-UV and mid-UV

    International Nuclear Information System (INIS)

    Ikebuchi, M.; Osmak, M.; Hill, C.

    1987-01-01

    Clones were isolated from U81 and N80 cells that were established by irradiation of Chinese hamster V79-M12G cells on a once a day schedule with 81 and 80 fractions of 6 J m/sup -2/ far-UV and 150 Jm/sup -2/ mid-UV (UV-B), respectively. These clones were examined for UV sensitivity to cell lethality and induction of mutations at 6TG/sup r/ (resistance to 6-thioguanine) and Oua/sup R/ (resistance to ouabain) loci. Survival curves for these clones indicate that their UV sensitivities to lethality vary from that of M12G cells to that of U81 and N80 parental cells. Clones also show heterogeneity for mutability to mid-UV: For induction of 6TG/sup r/, for example, non-mutable (U814), hypomutable (U815) and hypermutable (U811) were isolated from U81 cells. The authors are investigating by chromosome analysis and repair experiments why resistance to far-UV and mid-UV cell killing in these cells appears to be induced but the resulting survivors have a heterogeneous response to mutation induction by further doses of UV light

  2. Lectin histochemistry as a tool to identify apoptotic cells in the seminiferous epithelium of Syrian hamster (Mesocricetus auratus) subjected to short photoperiod.

    Science.gov (United States)

    Seco-Rovira, V; Beltrán-Frutos, E; Ferrer, C; Sánchez-Huertas, M M; Madrid, J F; Saez, F J; Pastor, L M

    2013-12-01

    Lectins have been widely used to study the pattern of cellular glycoconjugates in numerous species. In the process of cellular apoptosis, it has been observed that changes occur in the membrane sugar sequences of these apoptotic cells. The aim of our work was to identify which lectins, out of an extensive battery of the same (PNA, SBA, HPA, LTA, Con-A, UEA-I, WGA, DBA, MAA, GNA, AAA, SNA), show affinity for germinal cells in apoptosis, at what stage of cell death they do so and in which germinal cell types they can be detected. For this, we studied testis sections during testicular regression in Syrian hamster (Mesocricetus auratus) subjected to short photoperiod. Several lectins showed an affinity for the glycoconjugate residues of germ cells in apoptosis: Gal β1,3-GalNAcα1, α-d-mannose, N-acetylgalactosamine and l-fucose. Furthermore, lectin specificity was observed for some specific germinal cells and in certain stages of apoptosis. It was also observed that one of these lectins (PNA) showed affinity for Sertoli cells undergoing apoptosis. Therefore, we conclude that the use of lectin histochemistry could be a very useful tool for studying apoptosis in the seminiferous epithelium because of the specificity shown towards germinal cells in pathological or experimentally induced epithelial depletion models. © 2013 Blackwell Verlag GmbH.

  3. Identification of potential biomarkers in donor cows for in vitro embryo production by granulosa cell transcriptomics

    DEFF Research Database (Denmark)

    Mazzoni, Gianluca; Salleh, Suraya M; Freude, Kristine

    2017-01-01

    The Ovum Pick Up-In vitro Production (OPU-IVP) of embryos is an advanced reproductive technology used in cattle production but the complex biological mechanisms behind IVP outcomes are not fully understood. In this study we sequenced RNA of granulosa cells collected from Holstein cows at oocyte......) involved in these mechanisms. We found a range of evidence that good IVP outcome is positively correlated with early follicular atresia. Furthermore we showed that high genetic index bulls can be used in breeding without reducing the IVP performances. These findings can contribute to the development......, TNFAIP6 and TXNDC11 were negatively associated while Mx1 and STC1 were positively associated with all IVP scores. Functional analysis highlighted a wide range of biological mechanisms including apoptosis, cell development and proliferation and four key upstream regulators (COX2, IL1, PRL, TRIM24...

  4. Stem cells, embryos, and the environment: a context for both science and ethics.

    Science.gov (United States)

    Towns, C R; Jones, D G

    2004-08-01

    Debate on the potential and uses of human stem cells tends to be conducted by two constituencies-ethicists and scientists. On many occasions there is little communication between the two, with the result that ethical debate is not informed as well as it might be by scientific insights. The aim of this paper is to highlight those scientific insights that may be of relevance for ethical debate. Environmental factors play a significant role in identifying stem cells and their various subtypes. Research related to the role of the microenvironment has led to emphasis upon "plasticity", which denotes the ability of one type of stem cell to undergo a transition to cells from other lineages. This could increase the value given to adult stem cells, in comparison with embryonic stem cell research. Any such conclusion should be treated with caution, however, since optimism of this order is not borne out by current research. The role of the environment is also important in distinguishing between the terms totipotency and pluripotency. We argue that blastocysts (early embryos) and embryonic stem cells are only totipotent if they can develop within an appropriate environment. In the absence of this, they are merely pluripotent. Hence, blastocysts in the laboratory are potentially totipotent, in contrast to their counterparts within the human body which are actually totipotent. This may have implications for ethical debate, suggesting as it does that arguments based on potential for life may be of limited relevance.

  5. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation.

    Science.gov (United States)

    Sun, Xiaofei; Park, Craig B; Deng, Wenbo; Potter, S Steven; Dey, Sudhansu K

    2016-04-01

    Embryo implantation requires that the uterus differentiate into the receptive state. Failure to attain uterine receptivity will impede blastocyst attachment and result in a compromised pregnancy. The molecular mechanism by which the uterus transitions from the prereceptive to the receptive stage is complex, involving an intricate interplay of various molecules. We recently found that mice with uterine deletion ofMsxgenes (Msx1(d/d)/Msx2(d/d)) are infertile because of implantation failure associated with heightened apicobasal polarity of luminal epithelial cells during the receptive period. However, information on Msx's roles in regulating epithelial polarity remains limited. To gain further insight, we analyzed cell-type-specific gene expression by RNA sequencing of separated luminal epithelial and stromal cells by laser capture microdissection fromMsx1(d/d)/Msx2(d/d)and floxed mouse uteri on d 4 of pseudopregnancy. We found that claudin-1, a tight junction protein, and small proline-rich (Sprr2) protein, a major component of cornified envelopes in keratinized epidermis, were substantially up-regulated inMsx1(d/d)/Msx2(d/d)uterine epithelia. These factors also exhibited unique epithelial expression patterns at the implantation chamber (crypt) inMsx1(f/f)/Msx2(f/f)females; the patterns were lost inMsx1(d/d)/Msx2(d/d)epithelia on d 5, suggesting important roles during implantation. The results suggest thatMsxgenes play important roles during uterine receptivity including modulation of epithelial junctional activity.-Sun, X., Park, C. B., Deng, W., Potter, S. S., Dey, S. K. Uterine inactivation of muscle segment homeobox (Msx) genes alters epithelial cell junction proteins during embryo implantation. © FASEB.

  6. Development to term of sheep embryos reconstructed after inner cell mass/trophoblast exchange.

    Science.gov (United States)

    Loi, Pasqualino; Galli, Cesare; Lazzari, Giovanna; Matsukawa, Kazutsugu; Fulka, Josef; Goeritz, Frank; Hildebrandt, Thomas B

    2018-04-13

    Here we report in vitro and term development of sheep embryos after the inner cell mass (ICM) from one set of sheep blastocysts were injected into the trophoblast vesicles of another set. We also observed successful in vitro development of chimeric blastocysts made from sheep trophoblast vesicles injected with bovine ICM. First, we dissected ICMs from 35 sheep blastocysts using a stainless steel microblade and injected them into 29 re-expanded sheep trophoblastic vesicles. Of the 25 successfully micromanipulated trophoblastic vesicles, 15 (51.7%) re-expanded normally and showed proper ICM integration. The seven most well reconstructed embryos were transferred for development to term. Three ewes receiving manipulated blastocysts were pregnant at day 45 (42.8%), and all delivered normal offspring (singletons, two females and one male, average weight: 3.54 ± 0.358 kg). Next, we monitored in vitro development of sheep trophoblasts injected with bovine ICMs. Of 17 injected trophoblastic vesicles, 10 (58.8%) re-expanded after 4 h in culture, and four (40%) exhibited integrated bovine ICM. Our results indicate that ICM/trophoblast exchange is feasible, allowing full term development with satisfactory lambing rate. Therefore, ICM exchange is a promising approach for endangered species conservation.

  7. Essential role of chromatin remodeling protein Bptf in early mouse embryos and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Joseph Landry

    2008-10-01

    Full Text Available We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor, the largest subunit of NURF (Nucleosome Remodeling Factor in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf(-/- embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf(-/- embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo.

  8. Gamma-ray induced DNA breaks and repair studied by immuno-labelling of poly(ADP-ribose) polymerase (PARP) in chinese hamster ovary cells (CHO)

    International Nuclear Information System (INIS)

    Bidon, N.; Noel, G.; Averbeck, D.; Varlet, P.; Salamero, J.; DeMurcia, G.

    1998-01-01

    The poly(ADP-ribose)polymerase is a nuclear ubiquitous enzyme capable of binding to DNA breaks. Chinese hamster ovary cells were (CHO-K1) cultured on slides and γ-irradiated ( 137 Cs) at a high (12.8 Gy/min) or medium dose rate (5 Gy/min), and immuno-labelling against (ADP-ribose) polymers immediately or three hours after irradiation. Quantification and localisation of γ-ray induced breaks was performed by confocal microscopy. The results show a dose effect relationship, a dose-rate effect and the signal disappearance after 3 hours at 37 deg.C. The presence of PARP activity appears to reflect γ-rays induced DNA fragmentation. (authors)

  9. Control of the proportion of inner cells by asymmetric divisions and the ensuing resilience of cloned rabbit embryos

    Science.gov (United States)

    Duranthon, Véronique

    2018-01-01

    ABSTRACT Mammalian embryo cloning by nuclear transfer has a low success rate. This is hypothesized to correlate with a high variability of early developmental steps that segregate outer cells, which are fated to extra-embryonic tissues, from inner cells, which give rise to the embryo proper. Exploring the cell lineage of wild-type embryos and clones, imaged in toto until hatching, highlights the respective contributions of cell proliferation, death and asymmetric divisions to phenotypic variability. Preferential cell death of inner cells in clones, probably pertaining to the epigenetic plasticity of the transferred nucleus, is identified as a major difference with effects on the proportion of inner cell. In wild type and clones, similar patterns of outer cell asymmetric divisions are shown to be essential to the robust proportion of inner cells observed in wild type. Asymmetric inner cell division, which is not described in mice, is identified as a regulator of the proportion of inner cells and likely gives rise to resilient clones. PMID:29567671

  10. Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo.

    Science.gov (United States)

    Sepúlveda-Ramírez, Silvia P; Toledo-Jacobo, Leslie; Henson, John H; Shuster, Charles B

    2018-05-15

    In the sea urchin embryo, gastrulation is characterized by the ingression and directed cell migration of primary mesenchyme cells (PMCs), as well as the primary invagination and convergent extension of the endomesoderm. Like all cell shape changes, individual and collective cell motility is orchestrated by Rho family GTPases and their modulation of the actomyosin cytoskeleton. And while endomesoderm specification has been intensively studied in echinoids, much less is known about the proximate regulators driving cell motility. Toward these ends, we employed anti-sense morpholinos, mutant alleles and pharmacological inhibitors to assess the role of Cdc42 during sea urchin gastrulation. While inhibition of Cdc42 expression or activity had only mild effects on PMC ingression, PMC migration, alignment and skeletogenesis were disrupted in the absence of Cdc42, as well as elongation of the archenteron. PMC migration and patterning of the larval skeleton relies on the extension of filopodia, and Cdc42 was required for filopodia in vivo as well as in cultured PMCs. Lastly, filopodial extension required both Arp2/3 and formin actin-nucleating factors, supporting models of filopodial nucleation observed in other systems. Together, these results suggest that Cdc42 plays essential roles during PMC cell motility and organogenesis. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Self-Organization of Genome Expression from Embryo to Terminal Cell Fate: Single-Cell Statistical Mechanics of Biological Regulation

    Directory of Open Access Journals (Sweden)

    Alessandro Giuliani

    2017-12-01

    Full Text Available A statistical mechanical mean-field approach to the temporal development of biological regulation provides a phenomenological, but basic description of the dynamical behavior of genome expression in terms of autonomous self-organization with a critical transition (Self-Organized Criticality: SOC. This approach reveals the basis of self-regulation/organization of genome expression, where the extreme complexity of living matter precludes any strict mechanistic approach. The self-organization in SOC involves two critical behaviors: scaling-divergent behavior (genome avalanche and sandpile-type critical behavior. Genome avalanche patterns—competition between order (scaling and disorder (divergence reflect the opposite sequence of events characterizing the self-organization process in embryo development and helper T17 terminal cell differentiation, respectively. On the other hand, the temporal development of sandpile-type criticality (the degree of SOC control in mouse embryo suggests the existence of an SOC control landscape with a critical transition state (i.e., the erasure of zygote-state criticality. This indicates that a phase transition of the mouse genome before and after reprogramming (immediately after the late 2-cell state occurs through a dynamical change in a control parameter. This result provides a quantitative open-thermodynamic appreciation of the still largely qualitative notion of the epigenetic landscape. Our results suggest: (i the existence of coherent waves of condensation/de-condensation in chromatin, which are transmitted across regions of different gene-expression levels along the genome; and (ii essentially the same critical dynamics we observed for cell-differentiation processes exist in overall RNA expression during embryo development, which is particularly relevant because it gives further proof of SOC control of overall expression as a universal feature.

  12. Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

    International Nuclear Information System (INIS)

    Jung, Th.; Streffer, C.

    1992-01-01

    To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G 2 block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G 2 block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author)

  13. Effects of caffeine on protein phosphorylation and cell cycle progression in X-irradiated two-cell mouse embryos

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Th. (AFRC Institute of Animal Physiology and Genetics Research, Babraham (United Kingdom)); Streffer, C. (Essen Univ (Germany). Inst. fuer Medizinische Strahlenbiolgie)

    1992-08-01

    To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, the authors studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, caffeine was able to override the radiation induced G[sub 2] block and restored normal timing of these phosphorylation changes after X-irradiation. New additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced radiation induced G[sub 2] block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. (author).

  14. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research.

    Science.gov (United States)

    Manninen, Bertha Alvarez

    2008-01-31

    One argument used by detractors of human embryonic stem cell research (hESCR) invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event) as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical operation. Finally, I will end the

  15. Are human embryos Kantian persons?: Kantian considerations in favor of embryonic stem cell research

    Directory of Open Access Journals (Sweden)

    Manninen Bertha

    2008-01-01

    Full Text Available Abstract One argument used by detractors of human embryonic stem cell research (hESCR invokes Kant's formula of humanity, which proscribes treating persons solely as a means to an end, rather than as ends in themselves. According to Fuat S. Oduncu, for example, adhering to this imperative entails that human embryos should not be disaggregated to obtain pluripotent stem cells for hESCR. Given that human embryos are Kantian persons from the time of their conception, killing them to obtain their cells for research fails to treat them as ends in themselves. This argument assumes two points that are rather contentious given a Kantian framework. First, the argument assumes that when Kant maintains that humanity must be treated as an end in itself, he means to argue that all members of the species Homo sapiens must be treated as ends in themselves; that is, that Kant regards personhood as co-extensive with belonging to the species Homo sapiens. Second, the argument assumes that the event of conception is causally responsible for the genesis of a Kantian person and that, therefore, an embryo is a Kantian person from the time of its conception. In this paper, I will present challenges against these two assumptions by engaging in an exegetical study of some of Kant's works. First, I will illustrate that Kant did not use the term "humanity" to denote a biological species, but rather the capacity to set ends according to reason. Second, I will illustrate that it is difficult given a Kantian framework to denote conception (indeed any biological event as causally responsible for the creation of a person. Kant ascribed to a dualistic view of human agency, and personhood, according to him, was derived from the supersensible capacity for reason. To argue that a Kantian person is generated due to the event of conception ignores Kant's insistence in various aspects of his work that it is not possible to understand the generation of a person qua a physical

  16. Site-specific modification of genome with cell-permeable Cre fusion protein in preimplantation mouse embryo

    International Nuclear Information System (INIS)

    Kim, Kyoungmi; Kim, Hwain; Lee, Daekee

    2009-01-01

    Site-specific recombination (SSR) by Cre recombinase and its target sequence, loxP, is a valuable tool in genetic analysis of gene function. Recently, several studies reported successful application of Cre fusion protein containing protein transduction peptide for inducing gene modification in various mammalian cells including ES cell as well as in the whole animal. In this study, we show that a short incubation of preimplantation mouse embryos with purified cell-permeable Cre fusion protein results in efficient SSR. X-Gal staining of preimplantation embryos, heterozygous for Gtrosa26 tm1Sor , revealed that treatment of 1-cell or 2-cell embryos with 3 μM of Cre fusion protein for 2 h leads to Cre-mediated excision in 70-85% of embryos. We have examined the effect of the concentration of the Cre fusion protein and the duration of the treatment on embryonic development, established a condition for full term development and survival to adulthood, and demonstrated the germ line transmission of excised Gtrosa26 allele. Potential applications and advantages of the highly efficient technique described here are discussed.

  17. Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

    Science.gov (United States)

    Inoue, Kimiko; Ogura, Atsuo

    2013-01-01

    The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (Pcloning efficiency using SCNT. PMID:24146866

  18. Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Masunaga, Shin-ichiro [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst.; Keng, P.C.

    1996-11-01

    G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures. (author)

  19. Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro

    International Nuclear Information System (INIS)

    Masunaga, Shin-ichiro; Keng, P.C.

    1996-01-01

    G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures. (author)

  20. Inhibition of X-ray-induced potentially lethal damage (PLD) repair in aerobic plateau-phase Chinese hamster cells by misonidazole

    International Nuclear Information System (INIS)

    Brown, D.M.

    1984-01-01

    The effect of the 2-nitroimidazole radiosensitizer misonidazole (MISO) and the hydrophilic analog SR-2508 on the repair of X-ray-induced potentially lethal damage (PLD) was studied in plateau-phase Chinese Hamster ovary (HA-1) cells. It was found that although MISO does not radiosensitize aerobic cells, it inhibits the repair of PLD. However, under hypoxic conditions, MISO has no effect on PLD repair. The major portion of the inhibition of PLD repair in aerobic cells requires the presence of MISO only during irradiation; little or no additional inhibition occurs when MISO is present during the postirradiation repair period. Also, treatment of aerobic cells with 5 mM MISO for either 5 or 30 min prior to irradiation is equally inhibitory. This suggests that the presence of MISO in some way modifies the initial lesion under aerobic conditions since it does not increase cell killing as determined by immediate plating but inhibits subsequent repair. The inhibition is concentration dependent; 0.5 mM MISO inhibits PLD repair by one-half while 5-10 mM totally inhibits the repair measured 6 hr postirradiation. This phenomenon suggests that radiosensitization of tissue in vivo by MISO and other 2-nitroimidazoles may not be unequivocal proof of the presence of hypoxic cells

  1. [Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development].

    Science.gov (United States)

    Bellé, Robert; Le Bouffant, Ronan; Morales, Julia; Cosson, Bertrand; Cormier, Patrick; Mulner-Lorillon, Odile

    2007-01-01

    Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology

  2. Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells

    International Nuclear Information System (INIS)

    Jones, N.J.; Cox, R.; Thacker, J.

    1987-01-01

    The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC >> EMS > UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS > UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants. (Auth.)

  3. Beschermingsplan hamster 2005-2010

    NARCIS (Netherlands)

    Haye, la M.J.J.; Jansman, H.A.H.

    2005-01-01

    Alterra-Concept van het beschermingsplan hamster 2005-2010. De hamster is in het meest westelijke deel van het Europese verspreidingsgebied bedreigd. De kennis die in de afgelopen periode is opgedaan van de hamster en de maatregelen die in het veld zijn uitgevoerd vormen de basis voor dit tweede

  4. Mechanism of resistance of noncycling mammalian cells to 4'-(9-acridinylamino)methanesulfon-m-anisidide: comparison of uptake, metabolism, and DNA breakage in log- and plateau-phase Chinese hamster fibroblast cell cultures

    International Nuclear Information System (INIS)

    Robbie, M.A.; Baguley, B.C.; Denny, W.A.; Gavin, J.B.; Wilson, W.R.

    1988-01-01

    Resistance of noncycling cells to amsacrine (m-AMSA) has been widely reported and may limit the activity of this drug against solid tumors. The biochemical mechanism(s) for this resistance have been investigated using spontaneously transformed Chinese hamster fibroblasts (AA8 cells, a subline of Chinese hamster ovary-cells) in log- and plateau-phase spinner cultures. In early plateau phase most cells entered a growth-arrested state with a G1-G0 DNA content and showed a marked decrease in sensitivity to cytotoxicity induced by a 1-h exposure to m-AMSA or to its solid tumor-active analogue, CI-921. Studies with radiolabeled m-AMSA established that similar levels of drug were accumulated by log- and plateau-phase cells and that there was no significant drug metabolism in either of these cultures after 1 h. However, marked differences in sensitivity to m-AMSA-induced DNA breakage were observed using a fluorescence assay for DNA unwinding. Changes in sensitivity to DNA breakage occurred in parallel with changes in sensitivity to m-AMSA-induced cell killing. DNA breaks disappeared rapidly after drug removal (half-time approximately 4 min), suggesting that these lesions were probably mediated by DNA topoisomerase II. Resistance to m-AMSA may therefore be associated with changes in topoisomerase II activity in noncycling cells

  5. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology

    Directory of Open Access Journals (Sweden)

    Qisheng Zuo

    2016-06-01

    Full Text Available The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus. Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA recombination assay, T7 endonuclease I (T7EI assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%. Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens.

  6. Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

    LENUS (Irish Health Repository)

    Meleady, Paula

    2011-07-24

    Abstract Background The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. Results Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS\\/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. Conclusion These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

  7. An inhibitor of potentially lethal damage (PLD) repair reduces the frequency of γ-ray mutations in cultured Chinese hamster V79 cells

    International Nuclear Information System (INIS)

    Yokoiyama, A.; Kada, T.; Kuroda, Y.

    1992-01-01

    Cordycepin (3'-deoxyadenosine, 3 - dA) is an RNA antimetabolite and a radiosensitizer in cultured mammalian cells. In the present paper, the effects of 3'-dA on γ-ray-induced lethality and 6-thioguanine (6TG)-resistant mutations in cultured Chinese hamster V79 cells were examined. 3'-dA had the effect of sensitizing the lethality induced by γ-rays. The potentially lethal damage (PLD) repair produced by post-incubation cells in Hanks' solution after γ-irradiation was almost completely suppressed by 5x10 -5 M 3'-dA. When cells were irradiated with 10 Gy γ-rays and incubated with 3'-dA for 5 h, the frequency of 6TG-resistant mutations induced by γ-rays decreased to 1/6 of that of the irradiated cells incubated without 3'-dA. The decrease in the frequency of γ-ray-induced mutations was dependent on the length of incubation time with 3'-dA. It is suggested that the inhibition of PLD repair by 3'-dA may be that of error-prone repair. (author). 26 refs.; 5 figs

  8. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

    International Nuclear Information System (INIS)

    Nishijima, M.; Kuge, O.; Akamatsu, Y.

    1986-01-01

    The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32 Pi, the incorporation of 32 Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32 Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32 Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [ 3 H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U- 14 C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine

  9. Anthracyclines as radiosensitizers. A Cu(II) complex of a simpler analogue modifies DNA in Chinese Hamster V79 cells under low-dose γ radiation

    International Nuclear Information System (INIS)

    Saurabh Das; Mandal, P.C.

    2014-01-01

    Hydroxy-9,10-anthraquinones are structural analogues of anthracycline anticancer drugs showing similarity in physicochemical attributes, electrochemical behavior and biophysical interactions. 1,2-dihydroxy-9,10-anthraquinone (Q) and its complexes with Cu(II)/Ni(II) were studied for γ radiation induced modification of DNA in Chinese Hamster V79 cells. The amount of double stranded DNA remaining was ascertained by fluorometric analysis of DNA unwinding using ethidium bromide. Modification of double stranded DNA increased in the presence of Q and Cu(II)-Q when cells were irradiated (0-4.2 Gray). Ni(II)-Q was not that effective. Changing incubation time before recovery of DNA from cells there was evidence for DNA repair that was least for Cu(II)-Q treated cells. Minimum repair in case of Cu(II)-Q treated cells suggest the compound either assists radiation induced damage of agents responsible for repair or interacts with species like H 2 O 2 that assist in repair. Since a hydroxy-9,10-anthraquinone and its Cu(II) complex show radiosensitizing property, anthracyclines that contain a hydroxy-9,10-anthraquinone as the core moiety could also be tried as radiosensitizers in treating cancer. (author)

  10. Genome-wide host responses against infectious laryngotracheitis virus vaccine infection in chicken embryo lung cells

    Directory of Open Access Journals (Sweden)

    Lee Jeongyoon

    2012-04-01

    Full Text Available Abstract Background Infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1 infection causes high mortality and huge economic losses in the poultry industry. To protect chickens against ILTV infection, chicken-embryo origin (CEO and tissue-culture origin (TCO vaccines have been used. However, the transmission of vaccine ILTV from vaccinated- to unvaccinated chickens can cause severe respiratory disease. Previously, host cell responses against virulent ILTV infections were determined by microarray analysis. In this study, a microarray analysis was performed to understand host-vaccine ILTV interactions at the host gene transcription level. Results The 44 K chicken oligo microarrays were used, and the results were compared to those found in virulent ILTV infection. Total RNAs extracted from vaccine ILTV infected chicken embryo lung cells at 1, 2, 3 and 4 days post infection (dpi, compared to 0 dpi, were subjected to microarray assay using the two color hybridization method. Data analysis using JMP Genomics 5.0 and the Ingenuity Pathway Analysis (IPA program showed that 213 differentially expressed genes could be grouped into a number of functional categories including tissue development, cellular growth and proliferation, cellular movement, and inflammatory responses. Moreover, 10 possible gene networks were created by the IPA program to show intermolecular connections. Interestingly, of 213 differentially expressed genes, BMP2, C8orf79, F10, and NPY were expressed distinctly in vaccine ILTV infection when compared to virulent ILTV infection. Conclusions Comprehensive knowledge of gene expression and biological functionalities of host factors during vaccine ILTV infection can provide insight into host cellular defense mechanisms compared to those of virulent ILTV.

  11. X-ray induction of immortalization in primary rat embryo cells associated with and without tumorigenicity

    International Nuclear Information System (INIS)

    Sierra, E.; Oberley, L.W.; Guernsey, D.L.

    1985-01-01

    Cultures of primary rat embryo fibroblasts were irradiated with X-rays (3 Gy). After 14 days the majority of colonies in both irradiated and control plates had senesced. Surviving clones were ring isolated from irradiated and control plates and grown in culture. A phase of rapid proliferation after isolation was observed, followed by a decline (crisis) leading to senescence. Several clones from the irradiated plates were able to recover from this crisis and gave rise to continuous cell lines, while all colonies from control plates senesced. Three types of cells have been identified among the irradiated survivors: (1) immortal fully transformed, capable of growth in soft agar (Aga/sup +/) and tumor formation, (2) immortal normal, not able to grow in soft agar (Aga/sup -/) and nontumorigenic, and (3) immortal Aga/sup -/ cells which progressed to malignancy (Aga/sup +/, tumorigenicity) after further sub-culture. These data support the suggestion that X-rays can induce immortalization of mammalian cells in the absence of tumorigenicity, in addition to (and separate from) the fully tumorigenetic state

  12. Evidence for the induction of two types of potentially lethal damage after exposure of plateau phase Chinese hamster V79 cells to γ-rays

    International Nuclear Information System (INIS)

    Iliakis, G.

    1985-01-01

    The fixation of γ-rays induced potentially damage (PLD) caused after treatment either with β-araA or in medium made hypertonic by the addition of sodium chloride was studied in plateauphase chinese hamster V79 cells. Treatment with β-araA was found to affect a sector of PLD, the fixation of which specifically reduced the shoulder width of the survival curve. The effect was maximized when cell survival reached levels corresponding to an exponential line, with a slope similar to the final slope of the survival curve of untreated cells. This effect was achieved by a four hour treatment with β-araA at concentrations above 150 μM. Longer treatment times or incubation at higher β-araA concentrations did not significantly enhance the effect. Treatment in hypertonic medium, on the other hand, enhanced cell killing in a concentration dependent (NaCl-concentration) way and the survival reached values much lower than those corresponding to an exponential line. No indication for a plateau in the effect, indicating complete fixation of the sector of PLD that reacts sensitively to this treatment, was obtained. Boht the slope and the shoulder width of the survival curve were affected, the slope first being increaseed after short treatment times (up to 10 min), followed by a decrease in the shoulder width after longer treatment times (longer than 10 min). (orig./WL)

  13. Effects of agonist efficacy on desensitization of phosphoinositide hydrolysis mediated by m1 and m3 muscarinic receptors expressed in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Hu, J.; Wang, S.Z.; el-Fakahany, E.E.

    1991-01-01

    Muscarinic receptor agonist-induced desensitization of phosphoinositide (PI) hydrolysis and loss of receptors were studied in Chinese hamster ovary (CHO) cells transfected with the m1 and m3 muscarinic receptor genes. Long-term exposure to the full agonist carbamylcholine (CBC) resulted in a time-dependent attenuation of the maximal PI response and a decrease in agonist potency. This desensitization was accompanied by a parallel loss of maximal ligand binding without an alteration of the binding affinity. The time course of both receptor desensitization and down-regulation was similar in m1 and m3 CHO cells. The PI response to the partial agonist McN-A-343 (McN) in m1 cells was more sensitive to desensitization by CBC than the response to the latter agonist, and this desensitization was faster than receptor down-regulation. Desensitization of the PI response to McN was reflected as a decrease in the maximal response without a marked change in potency. McN induced slow desensitization of the PI response to CBC but a much faster desensitization of its own response. Our data provide evidence that although muscarinic agonist-induced desensitization of PI hydrolysis in CHO cells is due mainly to loss of receptors, there are other important factors which play a role in this process, e.g., receptor-effector uncoupling. The relative contribution of these different mechanisms depends on the efficacy of the agonists used for the receptor desensitization and activation steps

  14. Maize EMBRYO SAC family peptides interact differentially with pollen tubes and fungal cells.

    Science.gov (United States)

    Woriedh, Mayada; Merkl, Rainer; Dresselhaus, Thomas

    2015-08-01

    EMBRYO SAC1-4 (ES1-4) peptides belong to the defensin subgroup of cysteine-rich peptides known to mediate pollen tube burst in Zea mays (maize). ES1-4 are reported here to also be capable of inhibiting germination and growth of the maize fungal pathogens Fusarium graminearum and Ustilago maydis at higher concentrations. Dividing the peptides into smaller pieces showed that a 15-amino-acid peptide located in a highly variable loop region lacking similarity to other defensins or defensin-like peptides binds to maize pollen tube surfaces, causing swelling prior to burst. This peptide fragment and a second conserved neighbouring fragment showed suppression of fungal germination and growth. The two peptides caused swelling of fungal cells, production of reactive oxygen species, and finally the formation of big vacuoles prior to burst at high peptide concentration. Furthermore, peptide fragments were found to bind differently to fungal cells. In necrotrophic F. graminearum, a peptide fragment named ES-d bound only at cell surfaces whereas the peptide ES-c bound at cell surfaces and also accumulated inside cells. Conversely, in biotrophic U. maydis, both peptide fragments accumulated inside cells, but, if applied at higher concentration, ES-c but not ES-d accumulated mainly in vacuoles. Mapping of peptide interaction sites identified amino acids differing in pollen tube burst and fungal response reactions. In summary, these findings indicate that residues targeting pollen tube burst in maize are specific to the ES family, while residues targeting fungal growth are conserved within defensins and defensin-like peptides. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  15. The effect of γ-irradiation on the toxicity of malathion in V79 hamster cells and Molt-4 human lymphocytes

    International Nuclear Information System (INIS)

    Szekely, J.G.; Goodwin, M.; Delaney, S.

    1992-01-01

    There is a growing interest in irradiation of food and agricultural products for insect disinfestation, sprout inhibition, delayed ripening and the reduction of microbiological loads. Irradiation to a maximum dose of 10 kGy is recognized as safe by national and international regulatory agencies. To address the question, whether irradiation of pesticide residues might produce radiation products that were less or more toxic than the original pesticide, effects were observed of 10 kGy of γ-radiation on malathion as measured by sister-chromatid exchange (SCE), micronuclei formation, cell survival, growth rate and polyploid formation. No significant differences were found between effects of irradiated and unirradiated malathion on any of these end- points. Polyploid formation was the most dramatic effect of both irradiated and control malathion on V79 Chinese hamster cells. Cell survival, polyploid formation and growth rate were slightly better in cells treated with irradiated malathion. In Molt-4 human lymphocyte cell, micronuclei formation was not affected by unirradiated or irradiated malathion. Compared to malathion alone, the lack of such biological effects indicates that none of the presumed radiation-induced breakdown products increased or decreased the endpoints studied. The number of SCE was consistently, but not significantly, higher in cells treated with irradiated malathion. There were no significant differences in cell survival or micronucleus formation in the human lymphocyte cell line Molt-4 treated with irradiated or control malathion. Thus, the irradiation of the pesticide malathion to 10 kGy, a recommended upper dose for most food irradiations, does not significantly alter its toxicity in these in vitro systems. (author). 23 refs.; 4 figs.; 3 tabs

  16. Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

    Science.gov (United States)

    Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

    2012-01-01

    TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

  17. Monitoring utilizations of amino acids and vitamins in culture media and Chinese hamster ovary cells by liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Qiu, Jinshu; Chan, Pik Kay; Bondarenko, Pavel V

    2016-01-05

    Monitoring amino acids and vitamins is important for understanding human health, food nutrition and the culture of mammalian cells used to produce therapeutic proteins in biotechnology. A method including ion pairing reversed-phase liquid chromatography with tandem mass spectrometry was developed and optimized to quantify 21 amino acids and 9 water-soluble vitamins in Chinese hamster ovary (CHO) cells and culture media. By optimizing the chromatographic separation, scan time, monitoring time window, and sample preparation procedure, and using isotopically labeled (13)C, (15)N and (2)H internal standards, low limits of quantitation (≤0.054 mg/L), good precision (culture media. In a fed-batch process of manufacturing scale bioreactors, two distinguished trends for changes in amino acid concentrations were identified in response to feeding. Ten essential amino acids showed a zigzag pattern with maxima at the feeding days, and 9 non-essential amino acids displayed a smoothly changing profile as they were mainly products of cellular metabolism. Five of 9 vitamins accumulated continuously during the culture period, suggesting that they were fed in access. The method serves as an effective tool for the development and optimization of mammalian cell cultures. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Assessment of the developmental totipotency of neural cells in the cerebral cortex of mouse embryo by nuclear transfer

    Science.gov (United States)

    Yamazaki, Yukiko; Makino, Hatsune; Hamaguchi-Hamada, Kayoko; Hamada, Shun; Sugino, Hidehiko; Kawase, Eihachiro; Miyata, Takaki; Ogawa, Masaharu; Yanagimachi, Ryuzo; Yagi, Takeshi

    2001-01-01

    When neural cells were collected from the entire cerebral cortex of developing mouse fetuses (15.5–17.5 days postcoitum) and their nuclei were transferred into enucleated oocytes, 5.5% of the reconstructed oocytes developed into normal offspring. This success rate was th