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Sample records for hamster embryo cell

  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. 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

  3. 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

  4. 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

  5. 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.

  6. 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)

  7. 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.)

  8. 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

  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. 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.

  11. 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

  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. 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

  14. 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...

  15. 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

  16. 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)

  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. 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.

  19. 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.

  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. 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

  2. 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.)

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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...

  10. 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.

  11. 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

  12. 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)

  13. 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...

  14. 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

  15. 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

  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. 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

  18. 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.

  19. 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,

  20. Distinctive transforming genes in x-ray-transformed mammalian cells

    International Nuclear Information System (INIS)

    Borek, C.; Ong, A.; Mason, H.

    1987-01-01

    DNAs from hamster embryo cells and mouse C3H/10T1/2 cells transformed in vitro by x-irradiation into malignant cells transmit the radiation transformation phenotype by producing transformed colonies (transfectants) in two mouse recipient lines, the NIH 3T3 and C3H/101/2 cells, and in a rat cell line, the Rat-2 cells. DNAs from unirradiated cells or irradiated and visibly untransformed cells do not produce transformed colonies. The transfectant grow in agar and form tumors in nude mice. Treatment of the DNAs with restriction endonucleases prior to transfection indicates that the same transforming gene (oncogene) is present in each of the transformed mouse cells and is the same in each of the transformed hamster cells. Southern blot analysis of 3T3 or Rat-2 transfectants carrying oncogenes from radiation-transformed C3H/10T1/2 or hamster cells indicates that the oncogenes responsible for the transformation of 3T3 cells are not the Ki-ras, Ha-ras, N-ras genes, nor are they neu, trk, raf, abl, or fms. The work demonstrates that DNAs from mammalian cells transformed into malignancy by direct exposure in vitro to radiation contain genetic sequences with detectable transforming activity in three recipient cell lines. The results provide evidence that DNA is the target of radiation carcinogenesis induced at a cellular level in vitro. The experiments indicate that malignant radiogenic transformation in vitro of hamster embryo and mouse C3H/10T1/2 cells involves the activation of unique non-ras transforming genes, which heretofore have not been described

  1. 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

  2. [Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro].

    Science.gov (United States)

    Brusentsev, E Iu; Igonina, T N; Amstislavskiĭ, S Ia

    2014-01-01

    This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been consideredas laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.

  3. 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...

  4. 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

  5. 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)

  6. 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

  7. 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

  8. 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

  9. 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)

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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)

  16. 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

  17. 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.

  18. 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.)

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. Growth-differentiation factor-8 (GDF-8 in the uterus: its identification and functional significance in the golden hamster

    Directory of Open Access Journals (Sweden)

    O Wai Sum

    2009-11-01

    Full Text Available Abstract Transforming growth factor-beta superfamily regulates many aspects of reproduction in the female. We identified a novel member of this family, growth-differentiation factor 8 (GDF-8 in the 72 h post coital uterine fluid of the golden hamster by proteomic techniques. Uterine GDF-8 mRNA decreased as pregnancy progressed while its active protein peaked at 72 h post coitus (hpc and thereafter stayed at a lower level. At 72 hpc, the GDF-8 transcript was localized to the endometrial epithelium while its protein accumulated in the stroma. Exogenous GDF-8 slowed down proliferation of primary cultures of uterine smooth muscle cells (SMC and endometrial epithelial cells (EEC. In addition, GDF-8 attenuated the release of LIF (leukaemia inhibiting factor by EEC. As for the embryo in culture, GDF-8 promoted proliferation of the trophotoderm (TM and hatching but discouraged attachment. Our study suggests that GDF-8 could regulate the behavior of preimplantation embryos and fine-tune the physiology of uterine environment during pregnancy.

  11. 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

  12. 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...

  13. 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

  14. [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.

  15. 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

  16. 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...

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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)

  3. 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.

  4. Histaminergic regulation of seasonal metabolic rhythms in Siberian hamsters.

    Science.gov (United States)

    I'anson, Helen; Jethwa, Preeti H; Warner, Amy; Ebling, Francis J P

    2011-06-01

    We investigated whether histaminergic tone contributes to the seasonal catabolic state in Siberian hamsters by determining the effect of ablation of histaminergic neurons on food intake, metabolic rate and body weight. A ribosomal toxin (saporin) conjugated to orexin-B was infused into the ventral tuberomammillary region of the hypothalamus, since most histaminergic neurons express orexin receptors. This caused not only 75-80% loss of histaminergic neurons in the posterior hypothalamus, but also some loss of other orexin-receptor expressing cells e.g. MCH neurons. In the long-day anabolic state, lesions produced a transient post-surgical decrease in body weight, but the hamsters recovered and maintained constant body weight, whereas weight gradually increased in sham-lesioned hamsters. VO(2) in the dark phase was significantly higher in the lesioned hamsters compared to shams, and locomotor activity also tended to be higher. In a second study in short days, sham-treated hamsters showed the expected seasonal decrease in body weight, but weight remained constant in the lesioned hamsters, as in the long-day study. Lesioned hamsters consumed more during the early dark phase and less during the light phase due to an increase in the frequency of meals during the dark and decreased meal size during the light, and their cumulative food intake in their home cages was greater than in the control hamsters. In summary, ablation of orexin-responsive cells in the posterior hypothalamus blocks the short-day induced decline in body weight by preventing seasonal hypophagia, evidence consistent with the hypothesis that central histaminergic mechanisms contribute to long-term regulation of body weight. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. 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

  6. 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

  7. 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

  8. 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)

  9. 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.

  10. 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

  11. 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...

  12. 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....

  13. 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.

  14. 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.

  15. 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)

  16. 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...

  17. Effects of hyperthermia on the hamster immune system

    International Nuclear Information System (INIS)

    Gangavalli, R.; Cain, C.A.; Tompkins, W.A.F.

    1984-01-01

    In previous studies, the authors have shown that hyperthermia can enhance antibody-complement chytotoxicity of hamster and human tumor cells. Moreover, whole body microwave exposure of hamsters resulted in activation of peritoneal macrophages to a viricidal state and transient suppression of natural killer (NK) cell activity. In this study, the authors compare the effects of whole body heating by microwaves or by an environmental chamber (hot air) on the hamster immune system. Microwave exposure (25mW/cm/sup 2/; 1 hr) caused viricidal activation of peritoneal macrophages which resulted in restriction of vaccinia and vesicular stomatitis virs (VSV) growth. However, heating in an environmental chamber (41 0 C; 1 hr) did not activate macrophages to a viricidal state. Both microwave and hot air hyperthermia caused significant augmentation of antibody producing spleen cell response to sheep red blood cells (SRBC), using the Jerne hymolytic plaque assay, four days post exposure and immunization with SRBC. Natural killer spleen cell cytotoxicity was suppressed by microwave and hot air hyperthermia showing that NK lymphocytes are extremely sensitive to changes in temperature. These alterations in cellular immune response due to hyperthermia could be of significance in treatment of tumors and viral infections

  18. Effects of light deprivation on prolactin regulation in the Golden Syrian hamster

    International Nuclear Information System (INIS)

    Massa, J.S.

    1986-01-01

    Pineal-mediated depressions in prolactin cell activity after light deprivation were studied in the male and female Golden Syrian hamster. Prolactin cell activity was determined by measuring radioimmunoassayable prolactin, newly synthesized prolactin, newly synthesized prolactin and prolactin mRNA levels in the pituitary. Serum prolactin was also measured by radioimmunoassay. Use of the recombinant DNA plasmid, pPRL-1, which contains the rat prolactin complimentary DNA sequence, was validated in this dissertation for measuring prolactin mRNA in the hamster. Male Hamsters blinded for 11, 21, or 42 days showed significant and progressively greater declines in prolactin mRNA levels which were completely prevented by pinealectomy. Female hamsters blinded for 28 days, however, showed no such decreases in prolactin cell activity if they continued to display estrous cyclicity. After 12 weeks of blinding, females were acyclic and had dramatically depressed levels of prolactin cell activity. However, pinealectomy did not completely prevent this decline due to blinding unless the females continue to display estrous cyclicity. In ovariectomized females, blinding caused a decline in prolactin cell activity. In a separate study, significant changes in prolactin cell activity during the estrous cycle were seen in untreated normally cycling female hamsters. These changes in prolactin mRNA, prolactin synthesis, and radioimmunoassayable prolactin in the pituitary were measured in the morning, when, consistent with other reports, no differences in serum prolactin were observed

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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),

  6. Mouse Embryo Compaction.

    Science.gov (United States)

    White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

    2016-01-01

    Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

  7. 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...

  8. 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.

  9. 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

  10. Spermatogenesis is accelerated in the immature Djungarian and Chinese hamster and rat

    NARCIS (Netherlands)

    van Haaster, L. H.; de rooij, D. G.

    1993-01-01

    The rate of progression of spermatogenesis was studied in immature Djungarian and Chinese hamsters and Wistar rats by scoring the most advanced cell types present at various ages after birth. From 15 days of age onward, the most advanced cell types in the Djungarian hamsters were formed at a rate

  11. 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

  12. 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

  13. Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

    Directory of Open Access Journals (Sweden)

    M Imron

    2007-06-01

    Full Text Available In vivo embryo production was limited by number of donor, wide variability respond due to superovulation program and also immunoactifity of superovulation hormone (FSH. Splitting technology could be an alternative to increase the number of transferrable embryos into recipien cows. Splitting is done with cutting embryo becoming two equal pieces (called demi embrio base on ICM orientation. The objective of this research was to determine the viability of demi embryo obtained from embryo splitting of fresh and frozen thawed embryo. The results showed that demi embryos which performed blastocoel reexpansion 3 hours after embryo splitting using fresh and frozen thawed embryos were 76.9 and 76.2% respectively. Base on existention of inner cell mass (ICM, the number of demi embryos developed with ICM from fresh and frozen thawed embryos were not significantly different (90.6 and 85.7% respectively. The cell number of demi embryo from fresh embryos splitting was not different compared with those from frozen thawed embryos (36.1 and 35.9 respectively. These finding indicated that embryo splitting can be applied to frozen thawed embryos with certain condition as well as fresh embryos.

  14. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    Science.gov (United States)

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

  15. 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)

  16. 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...

  17. 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)

  18. 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...

  19. 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.

  20. Assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

    International Nuclear Information System (INIS)

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Miller, L.; Samuels, S.J.; Chang, R.J.; Overstreet, J.W.

    1988-01-01

    We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos

  1. 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.

  2. 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

  3. 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

  4. 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.)

  5. 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...

  6. 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

  7. 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.

  8. 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.

  9. 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....

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. 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)

  15. 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.

  16. 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

  17. 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.

  18. 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)

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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)

  5. Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.

    Science.gov (United States)

    Simmet, Kilian; Reichenbach, Myriam; Reichenbach, Horst-Dieter; Wolf, Eckhard

    2015-12-01

    Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. 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)

  7. 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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. Genetic changes in Mammalian cells transformed by helium cells

    Energy Technology Data Exchange (ETDEWEB)

    Durante, M.; Grossi, G. (Naples Univ. (Italy). Dipt. di Scienze Fisiche); Yang, T.C.; Roots, R. (Lawrence Berkeley Lab., CA (USA))

    1990-11-01

    Midterm Syrian Hamster embryo (SHE) cells were employed to study high LET-radiation induced tumorigenesis. Normal SHE cells (secondary passage) were irradiated with accelerated helium ions at an incident energy of 22 MeV/u (9--10 keV/{mu}m). Transformed clones were isolated after growth in soft agar of cells obtained from the foci of the initial monolayer plated postirradiation. To study the progression process of malignant transformation, the transformed clones were followed by monolayer subculturing for prolonged periods of time. Subsequently, neoplasia tests in nude mice were done. In this work, however, we have focused on karyotypic changes in the banding patterns of the chromosomes during the early part of the progressive process of cell transformation for helium ion-induced transformed cells. 26 refs., 5 figs., 2 tabs.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  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. 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)

  1. 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.

  2. 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

  3. 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)

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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...

  9. 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

  10. 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

  11. Mechanistic dissection of plant embryo initiation

    NARCIS (Netherlands)

    Radoeva, T.M.

    2016-01-01

    Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in

  12. 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.

  13. Interaction of x-rays and food pyrolysis products in producing oncogenic transformation in vitro

    International Nuclear Information System (INIS)

    Borek, C.; Ong, A.

    1981-01-01

    In recent years it has become evident from epidemiological and experimental data that a large number of environmental factors, including diet, play a role in modifying the incidence of cancer. Cell culture systems in which oncogenic transformation serves as an end point are powerful tools for evaluating these questions. Using such systems it has been shown recently that pyrolysis products from charred surfaces of broiled meat and fish can transform hamster embryo cells in vitro as well as produce tumors in the animal. Our studies in vitro have demonstrated the oncogenic potential of ionizing radiation in both hamster and human cells and have established in hamster cells the dose response relationship at doses ranging from 1 to 600 rad for x-rays and 0.1 to 150 rad for neutrons. The present work was aimed at evaluating whether there exists a cocarcinogenic interaction between a pyrolysis product and x-rays in their ability to transform hamster embryo cells in vitro. We have found that when cells are exposed to x-rays prior to treatment with the pyrolysis product there appears to be a synergistic interaction between the two agents in their ability to transform the cells

  14. 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...

  15. 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.)

  16. 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.

  17. 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

  18. 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)

  19. 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 ...

  20. 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)

  1. 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.

  2. 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

  3. 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.

  4. Identification of hamster inducible nitric oxide synthase (iNOS) promoter sequences that influence basal and inducible iNOS expression

    Science.gov (United States)

    Saldarriaga, Omar A.; Travi, Bruno L.; Choudhury, Goutam Ghosh; Melby, Peter C.

    2012-01-01

    IFN-γ/LPS-activated hamster (Mesocricetus auratus) macrophages express significantly less iNOS (NOS2) than activated mouse macrophages, which contributes to the hamster's susceptibility to intracellular pathogens. We determined a mechanism responsible for differences in iNOS promoter activity in hamsters and mice. The HtPP (1.2 kb) showed low basal and inducible promoter activity when compared with the mouse, and sequences within a 100-bp region (−233 to −133) of the mouse and hamster promoters influenced this activity. Moreover, within this 100 bp, we identified a smaller region (44 bp) in the mouse promoter, which recovered basal promoter activity when swapped into the hamster promoter. The mouse homolog (100-bp region) contained a cis-element for NF-IL-6 (−153/−142), which was absent in the hamster counterpart. EMSA and supershift assays revealed that the hamster sequence did not support the binding of NF-IL-6. Introduction of a functional NF-IL-6 binding sequence into the hamster promoter or its alteration in the mouse promoter revealed the critical importance of this transcription factor for full iNOS promoter activity. Furthermore, the binding of NF-IL-6 to the iNOS promoter (−153/−142) in vivo was increased in mouse cells but was reduced in hamster cells after IFN-γ/LPS stimulation. Differences in the activity of the iNOS promoters were evident in mouse and hamster cells, so they were not merely a result of species-specific differences in transcription factors. Thus, we have identified unique DNA sequences and a critical transcription factor, NF-IL-6, which contribute to the overall basal and inducible expression of hamster iNOS. PMID:22517919

  5. The effect of dietary vitamin A on NO2 exposure on the hamster lung

    International Nuclear Information System (INIS)

    Kim, J.C.

    1978-01-01

    The effect of dietary vitamin A and NO2 exposure on the hamster lung was evaluated by histopathology, electron microscopy, and thymidine uptake studies. Hamsters were maintained on deficient (0 micrograms), adequate (100 micrograms), and high (200 micrograms) dose levels of vitamin A while being exposed repeatedly to 10 ppm of NO2 for 5 hours once a week over an 8-week period. Hamsters of the deficient group exhibited clinical and morphologic changes characteristic of vitamin A deficiency. Animals maintained on adequate and high dose levels of vitamin A were not affected by vitamin A deficiency. Hypertrophy and hyperplasia of the epithelial cells of the terminal bronchiolar alveolar region of lungs of adequately and highly dosed animals were greater than those observed in the deficient animals, when NO2 exposure was given. However, the extent of the lesions observed in all three groups was less than that seen in normal hamsters given a single, 5-hour NO2 exposure. Ultrastructural changes observed in vitamin A-deficient hamsters exposed to NO2 were hypertrophy and hyperplasia of bronchiolar epithelial cells, diffuse loss of cilia, membrane damage, and mitochondrial damage manifested by calcium deposition. Tritiated thymidine uptake studies of lungs of animals exposed repeatedly revealed a rather erratic cell renewal pattern following NO2 exposure in comparison to the group of animals exposed singly

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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,...

  13. In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

    Science.gov (United States)

    Kelley, Rebecca L; Gardner, David K

    2017-05-01

    Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  14. 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

  15. 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.

  16. Expression of Aquaporins in Human Embryos and Potential Role of AQP3 and AQP7 in Preimplantation Mouse Embryo Development

    Directory of Open Access Journals (Sweden)

    Yun Xiong

    2013-05-01

    Full Text Available Background/Aims: Water channels, also named aquaporins (AQPs, play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

  17. Embryo density and medium volume effects on early murine embryo development.

    Science.gov (United States)

    Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

    1992-10-01

    One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

  18. 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

  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. Specificity of interaction between carcinogenic polynuclear aromatic hydrocarbons and nuclear proteins: widespread occurrence of a restricted pattern of histone-binding in intact cells

    International Nuclear Information System (INIS)

    MacLeod, M.C.; Pelling, J.C.; Slaga, T.J.; Nikbakht-Noghrei, P.A.; Mansfield, B.K.; Selkirk, J.K.

    1982-01-01

    Metabolic activation of benzo(a)pyrene [B(a)P] produces a number of potentially reactive metabolites. The endproducts of one metabolic pathway, 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydro-B(a)P (BPDE) are responsible for essentially all DNA adduct formation in animal cells treated with B(a)P, and a particular stereoisomer, designated (+)-anti-BPDE is thought to be the ultimate carcinogenic derivative of B(a)P. In hamster embryo cell nuclei treated with (+)-anti-BPDE, two of the histones of the nucleosomal core, H3 and H2A, are covalently modified, while the remaining core histones, H4 and H2B, are essentially unmodified. All four purified core histones, however, serve as targets. 7,12-dimethylbenz(a)anthracene and 3-methylcholanthrene show the same pattern of histone binding in hamster embryo cells. Treatment of mouse embryo cells with [ 3 H]-BPDE results in covalent binding of the hydrocarbon to histones H3 and H2A among the many cellular targets, while histones H2B and H4 are not bound. Similar binding patterns are seen in mouse embryo cells, a permanent murine, fibroblastic cell line, and a human mammary epithelial cell line, T47D, treated with [ 3 H]B(a)P. Again, the histones are unevenly labeled, displaying the H3 and H2A pattern. Histone-binding in the human cells may also be mediated by BPDE. Similar BPDE binding patterns were observed in other murine and human cell lines and in primary cultures of murine epidermal epithelial cells. The restriction of histone H2B and H4 binding appears to be general when intact cultured cells are studied. This specificity was not observed in a mixed reconstituted system in which rat liver microsomes were used to activate B(a)P. This finding reinforces reservations concerning the use of microsomal systems to probe the interactions of carcinogens with macromolecules and the relationships of adduct formation with the processes of carcinogenesis

  1. 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

  2. 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.

  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. 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.

  5. 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...

  6. Antigenic specificity and morphologic characteristics of Chlamydia trachomatis, strain SFPD, isolated from hamsters with proliferative ileitis.

    Science.gov (United States)

    Fox, J G; Stills, H F; Paster, B J; Dewhirst, F E; Yan, L; Palley, L; Prostak, K

    1993-10-01

    Profound diarrhea associated with proliferating intestinal cells containing intraepithelial campylobacter-like organisms (ICLO) occurs in a variety of mammalian hosts, particularly swine and hamsters. Recently, intracellular bacteria were isolated from proliferative intestinal tissue of hamsters and propagated in intestine cell line 407. Oral inoculation of hamsters with cell culture lysates containing these organisms reproduced the disease in susceptible hamsters. In the present study, an intracellular bacterium from the INT 407 cell line was shown by a variety of techniques to be a member of the genus Chlamydia and has been designated Chlamydia sp. strain SFPD. McCoy cells infected with Chlamydia sp. strain SFPD demonstrated bright fluorescent-stained intracytoplasmic inclusions when examined with fluorescein-labeled species-specific C. trachomatis monoclonal antibodies. The organism also reacted to fluorescein-labeled polyclonal but not monoclonal ICLO "omega" antisera. Ultrastructural examination of the Chlamydia sp. strain SFPD from McCoy cells revealed electrondense elementary bodies and a less electron-dense reticulate-like body that was circular; both features are consistent in morphology to developmental forms of Chlamydia and do not conform to ICLO morphology. Molecular studies, 16S ribosomal sequence analysis, and sequencing of the outer membrane protein confirmed that the isolate is a C. trachomatis closely related to the mouse pneumonitis strain of C. trachomatis.

  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. Autoradiography in fetal golden hamsters treated with tritiated diethylnitrosamine

    International Nuclear Information System (INIS)

    Reznik-Schueller, H.M.; Hague, B.F. Jr.

    1981-01-01

    Tritiated diethylnitrosamine was administered to female Syrian golden hamsters on each of the last 4 days (days 12-15) of pregnancy. The distribution of bound radioactivity was monitored by light microscopic autoradiography of fetal tracheas and livers, the placentas, and the maternal livers. In the trachea, the fetal target organ, bound radioactivity was restricted to the respiratory epithelium, where diethylnitrosamine-induced tracheal tumors arise. Mucous cells and nonciliated stem cells were identified as the principal sites of binding; other cell types within the tracheal epithelium contained only small amounts of bound radioactivity. The level of binding observed in the fetal trachea increased steadily from day 12 to day 15, which correlated well with the levels of differentiation of this tissue during this period. This observation also agrees with the previously reported observation that tumor incidence increases from 40 to 95% in Syrian golden hamsters between days 12 and 15

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. In vivo genetic toxicity studies in Chinese hamsters fed irradiated or unirradiated foodstuffs

    International Nuclear Information System (INIS)

    Altmann, H.

    1982-01-01

    Two in vivo genetic toxicity studies were performed in Chinese hamsters fed irradiated or unirradiated diets of chicken, fish or dates in order to detect possible mutagenic effects caused by irradiating these foodstuffs. The tests selected for study were: 1. Chromosomal analysis of bone narrow cells and 2. DNA metabolism in spleen cells. Chicken, fish and dates were irradiated with doses of 7, 2.5 and 1 kGy respectively. These investigations were subsequently extended to include the effects of irradiated dried onions, pulses and cocoa beans on DNA metabolism in Chinese hamster spleen cells only. Dried onions were irradiated with doses of 0.15, 9 and 15 kGy, pulses with 10 kGy and cocoa beans with 3.2 to 5 kGy. In addition, a fumigated cocoa bean group was included. No significant differences in chromosomal aberration rate were detected between groups fed irradiated or unirradiated diets. Dried dates, whether irradiated or not, showed some evidence of genetic toxicity in their effect on DNA metabolism in the spleen cells of Chinese hamsters. Both date diets caused more strand breaks DNA than are usual for Chinese hamster spleen cells, but DNA repair was not adversely affected. Chicken, both irradiated and unirradiated, was found to enhance replicative DNA synthesis but had no effect on the DNA repair process. Irradiated fish, however, caused enhanced DNA synthesis compared to unirradiated fish, but also had no adverse effect on DNA repair. Irradiated white beans also enhanced DNA synthesis compared to controls whereas unirradiated samples inhibited synthesis. (orig./MG)

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. [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.

  8. Zika virus infection of adult and fetal STAT2 knock-out hamsters.

    Science.gov (United States)

    Siddharthan, Venkatraman; Van Wettere, Arnaud J; Li, Rong; Miao, Jinxin; Wang, Zhongde; Morrey, John D; Julander, Justin G

    2017-07-01

    Zika virus (ZIKV) infection was investigated in adult and fetal STAT2 knock-out (KO) hamsters. Subcutaneous injection of ZIKV of adults resulted in morbidity, mortality, and infection of the uterus, placenta, brain, spinal cord, and testicles, thus providing an opportunity to evaluate congenital ZIKV infection in a second rodent species besides mice. ZIKV-infected cells with morphologies of Sertoli cells and spermatogonia were observed in the testes, which may have implications for sexual transmission and male sterility. Neonates exposed as fetuses to ZIKV at 8 days post-coitus were not smaller than controls. Nevertheless, infectious virus and ZIKV RNA was detected in some, but not all, placentas and fetal brains of KO hamsters. STAT2 KO hamsters may be useful for addressing sexual transmission, pathogenesis, routes of fetal infection, and neurological disease outcomes, and may also be used in antiviral or vaccine studies to identify intervention strategies. Copyright © 2017. Published by Elsevier Inc.

  9. Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus

    Directory of Open Access Journals (Sweden)

    Daniele Freitas Henriques

    2012-08-01

    Full Text Available Rocio virus (ROCV is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus. The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR. The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC. ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

  10. Electronegative LDL is linked to high-fat, high-cholesterol diet-induced nonalcoholic steatohepatitis in hamsters.

    Science.gov (United States)

    Lai, Yu-Sheng; Yang, Tzu-Ching; Chang, Po-Yuan; Chang, Shwu-Fen; Ho, Shu-Li; Chen, Hui-Ling; Lu, Shao-Chun

    2016-04-01

    The pathogenesis of nonalcoholic steatohepatitis (NASH), like that of atherosclerosis, involves lipid accumulation, inflammation and fibrosis. Recent studies suggest that oxidized LDL (oxLDL) may be a risk factor for NASH, but oxLDL levels were not directly measured in these studies. The aim of this study was to examine whether there was an association between electronegative LDL [LDL(-)], a mildly oxLDL found in the blood, and the development of NASH using two animal models. Golden Syrian hamsters and C57BL/6 mice were fed a high-fat, high-cholesterol (HFC) diet for 6 or 12weeks, then liver lipid and histopathology, plasma lipoprotein profile and LDL(-) levels were examined. The HFC-diet-fed hamsters and mice had similar levels of hepatic lipid but different histopathological changes, with microvesicular steatosis, hepatocellular hypertrophy, inflammation and bridging fibrosis in the hamsters, but only in mild steatohepatitis with low inflammatory cell infiltration in the mice. It also resulted in a significant increase in plasma levels of LDL cholesterol and LDL(-) in hamsters, but only a slight increase in mice. Moreover, enlarged Kupffer cells, LDL(-) and accumulation of unesterified cholesterol were detected in the portal area of HFC-diet-fed hamsters, but not HFC-diet-fed mice. An in vitro study showed that LDL(-) from HFC-diet-fed hamsters induced TNF-α secretion in rat Kupffer cell through a LOX-1-dependent pathway. Our results strongly suggest that LDL(-) is one of the underlying causes of hepatic inflammation and plays a critical role in the development of NASH. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. 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

  12. Developmental competence of porcine chimeric embryos produced by aggregation

    DEFF Research Database (Denmark)

    Li, Juan; Jakobsen, Jannik E.; Xiong, Qiang

    2015-01-01

    The purpose of our study was to compare the developmental competence and blastomere allocation of porcine chimeric embryos formed by micro-well aggregation. Chimeras were created by aggregating either two blastomeres originating from 2-cell embryos or two whole embryos, where embryos were produced...... either by parthenogenetic activation (PA) or handmade cloning (HMC). Results showed that the developmental competence of chimeric embryos, evaluated based on their blastocyst rate and total cell number per blastocyst, was increased when two whole 2-cell stage embryos (PA or HMC) were aggregated....... In comparison, when two blastomeres were aggregated, the developmental competence of the chimeric embryos decreased if the blastomeres were either from PA or from HMC embryos, but not if they were from different sources, i.e. one PA and one HMC blastomere. To evaluate the cell contribution in embryo formation...

  13. 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...

  14. 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.

  15. 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

  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. 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. 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)

  19. 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.

  20. 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...

  1. Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

    Science.gov (United States)

    Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

    2012-07-01

    To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

  2. 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)

  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. 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)

  5. Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system.

    Science.gov (United States)

    Ikeda, Shuntaro; Sugimoto, Miki; Kume, Shinichi

    2018-04-13

    Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P lipofection', may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

  6. 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

  7. Diet-induced metabolic hamster model of nonalcoholic fatty liver disease

    Directory of Open Access Journals (Sweden)

    Bhathena J

    2011-06-01

    Full Text Available Jasmine Bhathena, Arun Kulamarva, Christopher Martoni, Aleksandra Malgorzata Urbanska, Meenakshi Malhotra, Arghya Paul, Satya PrakashBiomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, Montreal, Québec, CanadaBackground: Obesity, hypercholesterolemia, elevated triglycerides, and type 2 diabetes are major risk factors for metabolic syndrome. Hamsters, unlike rats or mice, respond well to diet-induced obesity, increase body mass and adiposity on group housing, and increase food intake due to social confrontation-induced stress. They have a cardiovascular and hepatic system similar to that of humans, and can thus be a useful model for human pathophysiology.Methods: Experiments were planned to develop a diet-induced Bio F1B Golden Syrian hamster model of dyslipidemia and associated nonalcoholic fatty liver disease in the metabolic syndrome. Hamsters were fed a normal control diet, a high-fat/high-cholesterol diet, a high-fat/high-cholesterol/methionine-deficient/choline-devoid diet, and a high-fat/high-cholesterol/choline-deficient diet. Serum total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glucose, atherogenic index, and body weight were quantified biweekly. Fat deposition in the liver was observed and assessed following lipid staining with hematoxylin and eosin and with oil red O.Results: In this study, we established a diet-induced Bio F1B Golden Syrian hamster model for studying dyslipidemia and associated nonalcoholic fatty liver disease in the metabolic syndrome. Hyperlipidemia and elevated serum glucose concentrations were induced using this diet. Atherogenic index was elevated, increasing the risk for a cardiovascular event. Histological analysis of liver specimens at the end of four weeks showed increased fat deposition in the liver of animals fed

  8. Manipulating early pig embryos.

    Science.gov (United States)

    Niemann, H; Reichelt, B

    1993-01-01

    On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a long-term preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demi-embryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been born upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind

  9. 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

  10. 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

  11. 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)

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Die Behandlung menschliches Embryos und Menschenwurde

    OpenAIRE

    Matsui, Fumio

    2002-01-01

    We are confronted with an old and new problem, which has come up with the progress of modern biotechnologies: what is a life or when does a life begin? The expectation of order-made medicine has build up since the discovery of Embryo Stem cell called "a dream master cell", while there is any condemnation against the destruction of human embryo in order to gain it. It is a question whether a human embryo is a human being in the world. Human dignity(=HD) is a principle that keeps human embryos ...

  17. 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

  18. 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

  19. 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

  20. Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).

    Science.gov (United States)

    Correia, Sandra M; Canhoto, Jorge M

    2010-06-01

    The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.

  1. Hypothalamic ventricular ependymal thyroid hormone deiodinases are an important element of circannual timing in the Siberian hamster (Phodopus sungorus.

    Directory of Open Access Journals (Sweden)

    Annika Herwig

    Full Text Available Exposure to short days (SD induces profound changes in the physiology and behaviour of Siberian hamsters, including gonadal regression and up to 30% loss in body weight. In a continuous SD environment after approximately 20 weeks, Siberian hamsters spontaneously revert to a long day (LD phenotype, a phenomenon referred to as the photorefractory response. Previously we have identified a number of genes that are regulated by short photoperiod in the neuropil and ventricular ependymal (VE cells of the hypothalamus, although their importance and contribution to photoperiod induced physiology is unclear. In this refractory model we hypothesised that the return to LD physiology involves reversal of SD expression levels of key hypothalamic genes to their LD values and thereby implicate genes required for LD physiology. Male Siberian hamsters were kept in either LD or SD for up to 39 weeks during which time SD hamster body weight decreased before increasing, after more than 20 weeks, back to LD values. Brain tissue was collected between 14 and 39 weeks for in situ hybridization to determine hypothalamic gene expression. In VE cells lining the third ventricle, expression of nestin, vimentin, Crbp1 and Gpr50 were down-regulated at 18 weeks in SD photoperiod, but expression was not restored to the LD level in photorefractory hamsters. Dio2, Mct8 and Tsh-r expression were altered by SD photoperiod and were fully restored, or even exceeded values found in LD hamsters in the refractory state. In hypothalamic nuclei, expression of Srif and Mc3r mRNAs was altered at 18 weeks in SD, but were similar to LD expression values in photorefractory hamsters. We conclude that in refractory hamsters not all VE cell functions are required to establish LD physiology. However, thyroid hormone signalling from ependymal cells and reversal of neuronal gene expression appear to be essential for the SD refractory response.

  2. 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.)

  3. 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

  4. 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.

  5. 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.

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

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    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.

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

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    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.

  8. Action of uranium on pre implanted mouse embryos

    International Nuclear Information System (INIS)

    Kundt, Miriam S.

    2001-01-01

    The cultured preimplantation embryos are normally employed to evaluate the effects of environmental pollutants specially metals. Embryos were obtained from hybrid females CBA x C57 Bl following induction of super ovulation. They were incubated from 1 cell stage during 120 hs. in M16 cultured medium. Three different experiments were carried out: A, B and C using uranyl nitrate UO 2 (NO 3 ) 2 6H 2 O as source of uranium. In experiment 'A' the embryos were cultivated in the same culture dish containing final U concentrations of 13, 26, 52, 104 and 208 μgU/ml. In experiment 'B' embryos in a one cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104 μgU/ml. After 24 hours those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions of uranyl nitrate were added, maintaining the same concentrations of the previous one. In experiment 'C' the embryos were cultivated containing final U concentrations of 26, 52 and 104 μgU/ml and they were transferred to another culture dish every day to which fresh solutions of uranyl nitrate were added. Different embryos parameters were analyzed: 1) Development grade; 2) Number of cell per embryo and metaphases index; and 3) Embryo ploidy. 1) Embryos were observed each 24 hs. to evaluate development grade: 2, 4 and 8 cell stage, morula, early -expanded- hatched blastocysts and atresic embryos. No significant differences were observed in the proportion of embryos arrested either in the one-cell or in the two cell stages in control culture medium regarding different concentrations of U, in a total of 4388 embryos analyzed. From 2 cell stage, moment that the embryo begins to synthesize its own ARNm, the delay in embryonic development increased dose dependent. On the other hand, the toxicological effects in the same concentration are increase from 'A' treatment to 'C' treatment. Embriotoxicology effects are evidenced by an increment in

  9. Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos

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    B Cisterna

    2009-08-01

    Full Text Available In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in premRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

  10. Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

    Science.gov (United States)

    Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

    2008-01-01

    In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

  11. Enhancement of NMRI Mouse Embryo Development In vitro

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    Abedini, F.

    2013-12-01

    Full Text Available Most of the systematic studies used in the development of human embryo culture media have been done first on mouse embryos. The general use of NMRI outbred mice is a model for toxicology, teratology and pharmacology. NMRI mouse embryo exhibit the two-cell block in vitro. The objective of this study was to evaluate and compare the effects of four kinds of culture media on the development of zygotes (NMRI after embryo vitrification. One-cell mouse embryos were obtained from NMRI mice after superovulation and mating with adult male NMRI mice. And then randomly divided into 4 groups for culture in four different cultures media including: M16 (A, DMEM/Ham, F-12 (B, DMEM/Ham's F-12 co-culture with Vero cells(C and DMEM/Ham's F-12 co-culture with MEF cells (D. Afterward all of the embryos were vitrified in EFS40 solution and collected. Results of our study revealed, more blastocysts significantly were developed with co-culture with MEF cells in DMEM/Ham's F-12 medium. More research needed to understand the effect of other components of culture medium, and co-culture on NMRI embryo development.

  12. 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.

  13. The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos.

    Science.gov (United States)

    Kim, Eun Young; Lee, Jun Beom; Park, Hyo Young; Jeong, Chang Jin; Riu, Key Zung; Park, Se Pill

    2011-06-01

    Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; Pculture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.

  14. 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).

  15. Secondhand smoke induces hepatic apoptosis and fibrosis in hamster fetus.

    Science.gov (United States)

    Huang, Chien-Wei; Horng, Chi-Ting; Huang, Chih-Yang; Cho, Ta-Hsiung; Tsai, Yi-Chang; Chen, Li-Jeng; Hsu, Tsai-Ching; Tzang, Bor-Show

    2016-09-01

    Secondhand smoke (SHS) is an important health issue worldwide. Inhaling SHS during pregnancy could cause abnormalities in the internal tissues of newborns, which may then impair fetal development and even cause severe intrauterine damage and perinatal death. However, the understanding of cytopathic mechanisms of SHS by maternal passive smoking on fetus liver during pregnancy is still limited. This study analyzed the effects of high-dose SHS (SHSH) on fetus liver using a maternal passive smoking animal model. Experiments showed that hepatic matrix metalloproteinase-9 activity and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling-positive cells were significantly increased in livers from fetuses of hamsters treated with SHSH. Similarly, expressions of both extrinsic and intrinsic apoptotic molecules were significantly higher in livers from fetuses of hamsters exposed to SHSH. Additionally, significantly increased inflammatory proteins, including transforming growth factor β, inducible nitric oxide synthase, and interleukin 1β, and fibrotic signaling molecules, including phosphorylated Smad2/3, SP1, and α-smooth muscle actin, were observed in the fetus livers from hamsters treated with SHSH. This study revealed that SHSH not only increased apoptosis through intrinsic and extrinsic pathways in the livers of fetuses from hamsters exposed to SHSH but also augmented hepatic fibrosis via Smad2/3 signaling. © The Author(s) 2015.

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

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    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.

  17. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    Science.gov (United States)

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  18. 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

  19. 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.

  20. 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.

  1. Effect of the microenvironment and embryo density on developmental characteristics and gene expression profile of bovine preimplantative embryos cultured in vitro.

    Science.gov (United States)

    Hoelker, Michael; Rings, Franka; Lund, Qamaruddin; Ghanem, Nasser; Phatsara, Chirawath; Griese, Josef; Schellander, Karl; Tesfaye, Dawit

    2009-03-01

    The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos.

  2. 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

  3. 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

  4. Test for antioxidant ability by scavenging long-lived mutagenic radicals in mammalian cells and by blood test with intentional radicals: an application of gallic acid

    International Nuclear Information System (INIS)

    Kumagai, Jun; Kawaura, Tomoko; Miyazaki, Tetsuo; Prost, Michel; Prost, Emmanuelle; Watanabe, Masami; Quetin-Leclercq, J.Joeelle

    2003-01-01

    Antioxidant ability of gallic acid (GA) are determined both by electron spin resonance measurement of long-lived radicals produced in γ-ray irradiated Syrian golden hamster embryo cells with GA and by hemolysis measurement with GA when blood cells are submitted to radicals. Scavenging properties of GA are determined by the reaction rate constant with long-lived mutagenic radicals in the cells while the blood test allows to analyze the global effects of this compound: radical scavenger+metal ion chelator+regeneration of intra- and extra-cellular antioxidant

  5. Elk3 from hamster-a ternary complex factor with strong transcriptional repressor activity

    DEFF Research Database (Denmark)

    Hjortoe, G.M.; Weilguny, D.; Willumsen, Berthe Marie

    2005-01-01

    the transcription of genes that are activated during entry into G1. We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c......-fos promoter, Elk3 from CHO cells seems to inhibit other promoters controlling expression of proteins involved in G1/S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicate that hamster Elk3...

  6. Untwisting the Caenorhabditis elegans embryo

    Science.gov (United States)

    Christensen, Ryan Patrick; Bokinsky, Alexandra; Santella, Anthony; Wu, Yicong; Marquina-Solis, Javier; Guo, Min; Kovacevic, Ismar; Kumar, Abhishek; Winter, Peter W; Tashakkori, Nicole; McCreedy, Evan; Liu, Huafeng; McAuliffe, Matthew; Mohler, William; Colón-Ramos, Daniel A; Bao, Zhirong; Shroff, Hari

    2015-01-01

    The nematode Caenorhabditis elegans possesses a simple embryonic nervous system with few enough neurons that the growth of each cell could be followed to provide a systems-level view of development. However, studies of single cell development have largely been conducted in fixed or pre-twitching live embryos, because of technical difficulties associated with embryo movement in late embryogenesis. We present open-source untwisting and annotation software (http://mipav.cit.nih.gov/plugin_jws/mipav_worm_plugin.php) that allows the investigation of neurodevelopmental events in late embryogenesis and apply it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos. We also provide a tutorial describing how to use the software (Supplementary file 1) and a detailed description of the untwisting algorithm (Appendix). The detailed positional information we obtained enabled us to develop a composite model showing movement of these cells and neurites in an 'average' worm embryo. The untwisting and cell tracking capabilities of our method provide a foundation on which to catalog C. elegans neurodevelopment, allowing interrogation of developmental events in previously inaccessible periods of embryogenesis. DOI: http://dx.doi.org/10.7554/eLife.10070.001 PMID:26633880

  7. Untwisting the Caenorhabditis elegans embryo.

    Science.gov (United States)

    Christensen, Ryan Patrick; Bokinsky, Alexandra; Santella, Anthony; Wu, Yicong; Marquina-Solis, Javier; Guo, Min; Kovacevic, Ismar; Kumar, Abhishek; Winter, Peter W; Tashakkori, Nicole; McCreedy, Evan; Liu, Huafeng; McAuliffe, Matthew; Mohler, William; Colón-Ramos, Daniel A; Bao, Zhirong; Shroff, Hari

    2015-12-03

    The nematode Caenorhabditis elegans possesses a simple embryonic nervous system with few enough neurons that the growth of each cell could be followed to provide a systems-level view of development. However, studies of single cell development have largely been conducted in fixed or pre-twitching live embryos, because of technical difficulties associated with embryo movement in late embryogenesis. We present open-source untwisting and annotation software (http://mipav.cit.nih.gov/plugin_jws/mipav_worm_plugin.php) that allows the investigation of neurodevelopmental events in late embryogenesis and apply it to track the 3D positions of seam cell nuclei, neurons, and neurites in multiple elongating embryos. We also provide a tutorial describing how to use the software (Supplementary file 1) and a detailed description of the untwisting algorithm (Appendix). The detailed positional information we obtained enabled us to develop a composite model showing movement of these cells and neurites in an 'average' worm embryo. The untwisting and cell tracking capabilities of our method provide a foundation on which to catalog C. elegans neurodevelopment, allowing interrogation of developmental events in previously inaccessible periods of embryogenesis.

  8. 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.

  9. Nucleolar remodeling in nuclear transfer embryos

    DEFF Research Database (Denmark)

    Laurincik, Jozef; Maddox-Hyttel, Poul

    2007-01-01

    Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate the devel......Transcription of the ribosomal RNA (rRNA) genes occurs in the nucleolus and results in ribosome biogenesis. The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus serve to evaluate...... nucleoli are not apparent until the 5th cell cycle, whereas in somatic cell nuclear transfer embryos the functional nucleoli emerge already during the 3rd cell cycle. Intergeneric reconstructed embryos produced by the fusion of bovine differentiated somatic cell to a nonactivated ovine cytoplast fail...

  10. 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)

  11. 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

  12. 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.

  13. 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...

  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. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  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. 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.

  2. 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.

  3. 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

  4. 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

  5. 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...

  6. Modulating effects of the protease inhibitor Antipain on x-ray induced transformations

    International Nuclear Information System (INIS)

    Borek, C.; Miller, R.C.

    1979-01-01

    Protease inhibitors have been shown to inhibit the expression of mutations in bacteria and to inhibit the tumor-promoting effect of phorbol esters in mice. We have investigated the effect of the protease inhibitor Antipain on cell transformation by x-irradiation in two in vitro systems; namely short-term cultures of freshly explanted hamster embryo cells and in the 10T1/2 cell line derived and cloned from C3H mouse embryo

  7. 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

  8. 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.)

  9. 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)

  10. 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

  11. 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.

  12. Radiobiology

    International Nuclear Information System (INIS)

    Anon.

    1975-01-01

    The effects of metridazole and nitroimidazole on the survival time of cultured hamster cells following exposure to various doses and dose rates of 60 Co γ radiation or neutrons were studied. Both were found to increase the radiosensitivity of the cells. Data are included on the modifying effects of neutron spectra, energy levels, LET, OER, dose, and dose fractionation schedules on the γ and neutron sensitivity of cultured hamster cells. Studies on the sensitivity of cultured hamster cells and normal liver and hepatoma cells to hyperthermia and hypoxia, with and without the added effects of x irradiation showed that heat treatment at 43 0 C enhanced the radiosensitivity of the cells, with hypoxic cells being the most sensitive. A system was developed for the study of radioinduced carcinogenesis in cultured hamster embryo cells. Preliminary data are presented on the dose response relationships for transformation following exposure to x radiation or neutrons. (U.S.)

  13. 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.)

  14. 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

  15. 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

  16. 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...

  17. The induction by X-rays of chromosome aberrations in male guinea-pigs, golden hamsters and rabbits

    International Nuclear Information System (INIS)

    Cox, B.D.; Lyon, M.F.

    1975-01-01

    Translocations induced by X-rays in post-meiotic germ cells of male guinea-pigs, golden hamsters and rabbits were studied cytologically in the F 1 sons of the irradiated males. The percentage of spermatocytes displaying multivalent configurations varied with the translocation, but the average percentage appeared to depend on the species: fewer quadrivalents were observed in hamster than in guinea-pig heterozygotes and most were recorded for rabbit heterozygotes. Chain quadrivalents were more abundant than ring quadrivalents at meiosis for the guinea-pig and hamster in contrast to the mouse. Too few translocation heterozygotes were examined to determine which meiotic configuration was the more prevalent in the rabbit. In all three species, as in the mouse, translocations were found which caused male sterility, due to partial or complete failure of spermatogenesis, although most translocations caused semi-sterility. For these semi-sterile males both the frequency and time of embryonic death in the progeny appeared to be the same as in the mouse. It is concluded that similar types of chromosome aberrations are induced by X-rays in post-meiotic germ cells of male guinea-pigs, rabbits, golden hamsters and mice

  18. Temperature-sensitive host range mutants of herpes simplex virus type 2

    International Nuclear Information System (INIS)

    Koment, R.W.; Rapp, F.

    1975-01-01

    Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblast cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties

  19. 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.)

  20. 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

  1. 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.

  2. 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...

  3. Rape embryogenesis. III. Embryo development in time

    Directory of Open Access Journals (Sweden)

    Teresa Tykarska

    2014-01-01

    Full Text Available It was found that the growth curve of the rape embryo axis is of triple sigmoid type. Embryo growth occurs in 3 phases corresponding to 3 different periods of development. Phase I includes growth of the apical cell up to it's division into two layers of octants. Phase II comprises the increase of the spherical proembryo to the change of its symmetry from radial to bilateral. Phase III includes, growth of the embryo from the heart stage up to the end of embryogenesis. In each phase the relative growth rate increases drastically and then diminishes. The differences in growth intensity during the same phase are several-fold. The growth intensity maximum of the embryo axis occurs in phase II. The phasic growth intensity maxima occur: in phase I during apical cell elongation, :before its division, and in phases II and III in the periods of cell division ;growth in globular and torpedo-shaped -shaped embryos.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. Association of 239Pu with lysosomes in rat, Syrian hamster, and Chinese hamster liver as studied by carrier-free electrophoresis and electron microscopic autoradiography with 241Pu

    International Nuclear Information System (INIS)

    Seidel, A.; Krueger, E.W.; Wiener, M.; Hotz, G.; Balani, M.; Thies, W.G.

    1985-01-01

    The binding of injected monomeric plutonium in the liver of rats, Syrian hamsters, and Chinese hamsters (species which show profound differences in their ability to eliminate 239 Pu from the liver) was investigated by carrier-free electrophoresis using 239 Pu and electron microscopic autoradiography with 241 Pu. These studies are part of a program designed to obtain a better understanding of the mechanisms of the clearance of transuranium elements from liver of different mammals and man. Between 4 and 9 days after nuclide injection, a clear correlation between the majority of the 239 Pu and lysosomal enzymes was observed when the mitochondrial-lysosomal (ML) fraction of the livers was analyzed by carrier-free electrophoresis. In the two hamster species, a second 239 Pu peak exists from the beginning and increases with time to comprise 50% of the total radioactivity at later times. During electron microscopic examination 4 days after 241 Pu injection, beta tracks were frequently observed over globular structures resembling dense bodies in Chinese hamster liver. They were also observed frequently over chromatin-rich portions of the cell nuclei. These results, together with those from previous density gradient studies, show that lysosomes are the primary deposition site for 239 Pu in the liver cytoplasm of these three rodent species. The hypothesis of a morphologic transformation of these lysosomes with time in hamster liver and of rapid bulk exocytosis of the lysosomes in rats are still possible explanations for the extreme differences in the elimination among the three species

  15. Effect of embryo density on in vitro development and gene expression in bovine in vitro-fertilized embryos cultured in a microwell system.

    Science.gov (United States)

    Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Aikawa, Yoshio; Ohtake, Masaki; Matsuda, Hideo; Kobayashi, Shuji; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

    2013-01-01

    To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

  16. 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.

  17. Nucleolar ultrastructure in bovine nuclear transfer embryos

    DEFF Research Database (Denmark)

    Kanka, J; Smith, S D; Soloy, E

    1999-01-01

    in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

  18. 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

  19. Morphometric studies with attached mouse C3H/10T 1/2 cells

    International Nuclear Information System (INIS)

    Geard, C.R.; Harding, T.

    1981-01-01

    Studies of in vitro transformation using the Syrian hamster embryo cell system and the mouse C3H/10T 1/2 cell system form an integral part of this laboratory's activities. As part of the studies with the mouse cell line we have monitored the behavior of these cells in culture in order to ascertain those variables which might influence the expression of transformation. The study of transformed cells versus normal cells could lead to insight into an earlier definition of transformation that the clonal morphological change currently in use. This present report details the changes in cellular morphology with time in culture of normal mouse C3H/10T 1/2 cells from early passages (9 to 13) and x-ray transformed cells which have been maintained in culture for three years

  20. Production and purification of polyclonal anti-hamster ...

    African Journals Online (AJOL)

    . ... IgG showed high titer and high specificity in the designed ELISA. Purified antibody and its conjugation with HRP are used in research and diagnosis of hamster disease. Key words: Production, purification, hamster immunoglobulins.

  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. Surgical manipulation of mammalian embryos in vitro.

    Science.gov (United States)

    Naruse, I; Keino, H; Taniguchi, M

    1997-04-01

    Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

  3. 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.

  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. 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...

  6. [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.

  7. 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 ...

  8. Radioprotective effect of catecholamines on the cultured Chinese hamster fibroblasts

    International Nuclear Information System (INIS)

    Chirkov, Yu.Yu.; Malatsidze, M.A.; Sobolev, A.S.

    1985-01-01

    On cultivated in vitro Chinese hamster fibroblasts radioprotective properties of adrenaline, noradrenaline and isoproterenol in different concentrations are studied. Isoproterenol radiopreventive effect is clearly manifested with its concentration being 1x10 -8 M; adrenaline and noradrenaline are efficient in higher concentrations. Propranolol, blocking β-adrenergic receptors, completely presents radioprotective effect of catecholamines on the cells. β-adrenergic mechanism of catecholamine radioprotective effect on Mammalia cells is discussed

  9. 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.

  10. Analysis of compaction initiation in human embryos by using time-lapse cinematography.

    Science.gov (United States)

    Iwata, Kyoko; Yumoto, Keitaro; Sugishima, Minako; Mizoguchi, Chizuru; Kai, Yoshiteru; Iba, Yumiko; Mio, Yasuyuki

    2014-04-01

    To analyze the initiation of compaction in human embryos in vitro by using time-lapse cinematography (TLC), with the goal of determining the precise timing of compaction and clarifying the morphological changes underlying the compaction process. One hundred and fifteen embryos donated by couples with no further need for embryo-transfer were used in this study. Donated embryos were thawed and processed, and then their morphological behavior during the initiation of compaction was dynamically observed via time-lapse cinematography (TLC) for 5 days. Although the initiation of compaction occurred throughout the period from the 4-cell to 16-cell stage, 99 (86.1 %) embryos initiated compaction at the 8-cell stage or later, with initiation at the 8-cell stage being most frequent (22.6 %). Of these 99 embryos, 49.5 % developed into good-quality blastocysts. In contrast, of the 16 (13.9 %) embryos that initiated compaction prior to the 8-cell stage, only 18.8 % developed into good-quality blastocysts. Embryos that initiated compaction before the 8-cell stage showed significantly higher numbers of multinucleated blastomeres, due to asynchronism in nuclear division at the third mitotic division resulting from cytokinetic failure. The initiation of compaction primarily occurs at the third mitotic division or later in human embryos. Embryos that initiate compaction before the 8-cell stage are usually associated with aberrant embryonic development (i.e., cytokinetic failure accompanied by karyokinesis).

  11. Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

    Science.gov (United States)

    Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

    2010-08-01

    We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

  12. 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.

  13. Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation*

    Science.gov (United States)

    Uchida, Hiroshi; Maruyama, Tetsuo; Nishikawa-Uchida, Sayaka; Oda, Hideyuki; Miyazaki, Kaoru; Yamasaki, Akiko; Yoshimura, Yasunori

    2012-01-01

    Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin. PMID:22174415

  14. 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 the highest among all previous mouse cloning experiments that used somatic cells. Forty-four percent of live embryos at 10.5 days postcoitum were morphologically normal when premature and early-postmitotic neural cells from the ventricular side of the cortex were used. In contrast, the majority (95%) of embryos were morphologically abnormal (including structural abnormalities in the neural tube) when postmitotic-differentiated neurons from the pial side of the cortex were used for cloning. Whereas 4.3% of embryos cloned with ventricular-side cells developed into healthy offspring, only 0.5% of those cloned with differentiated neurons in the pial side did so. These facts seem to suggest that the nuclei of neural cells in advanced stages of differentiation had lost their developmental totipotency. The underlying mechanism for this developmental limitation could be somatic DNA rearrangements in differentiating neural cells. PMID:11698647

  15. 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).

  16. 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.

  17. Brachyury expression in tailless Molgulid ascidian embryos.

    Science.gov (United States)

    Takada, Norio; York, Jonathan; Davis, J Muse; Schumpert, Brenda; Yasuo, Hitoyoshi; Satoh, Nori; Swalla, Billie J

    2002-01-01

    The T-box transcription factor gene Brachyury is important for the differentiation of notochord in all chordates, including the ascidians Halocynthia roretzi and Ciona intestinalis. We isolated Brachyury from molgulid ascidians, which have evolved tailless larvae multiple times independently, and found the genes appear functional by cDNA sequence analyses. We then compared the expression of Mocu-Bra in tailed Molgula oculata embryos to two tailless species, Molgula occulta (Mocc-Bra) and Molgula tectiformis (Mt-Bra). Here we show that both tailless species express Brachyury in the notochord lineage during embryogenesis. Initial expression of Mocu-Bra is normal in tailed M. oculata embryos; 10 precursor notochord cells divide twice to result in 40 notochord cells that converge and extend to make a notochord down the center of the tail. In contrast, in tailless Molgula occulta, Mocc-Bra expression disappears prematurely, and there is only one round of division, resulting in 20 cells in the final notochord lineage that never converge or extend. In M. occulta x M. oculata hybrid embryos, expression of Mocu-Bra is prolonged, and the embryos form a tail with 20 notochord cells that converge and extend normally. However, in Molgula tectiformis, a different tailless ascidian, Mt-Bra was expressed only in the 10 notochord precursor cells, which never divide, converge, or extend. In summary, neither Brachyury function nor the early establishment of the notochord lineage appears to be impaired in tailless embryos. In light of these results, we are continuing to investigate how and why notochord development is lost in tailless molgulid ascidian embryos.

  18. Regional localization of suspensor mRNAs during early embryo development.

    Science.gov (United States)

    Weterings, K; Apuya, N R; Bi, Y; Fischer, R L; Harada, J J; Goldberg, R B

    2001-11-01

    We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/beta-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote.

  19. Regional Localization of Suspensor mRNAs during Early Embryo Development

    Science.gov (United States)

    Weterings, Koen; Apuya, Nestor R.; Bi, Yuping; Fischer, Robert L.; Harada, John J.; Goldberg, Robert B.

    2001-01-01

    We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/β-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote. PMID:11701878

  20. Membrane associated ion transport enzymes in normal and transformed fibroblasts and epithelial cells

    International Nuclear Information System (INIS)

    Borek, C.

    1982-01-01

    In an effort to evaluate membrane changes associated with neoplastic transformation of fibroblasts and epithelial cells by radiation and chemicals, alterations in membrane-associated (Na + + K + )-ATPase and 5'-nucleotidase activities were investigated. Cell cultures consisted of normal and radiation transformed hamster embryo fibroblasts (HE) and mouse C3H 10T 1/2 fibroblasts, normal and chemically transformed adult rat liver epithelial cells (ARL), as well as hepatocarcinoma cells induced by the liver transformants. Transformed fibroblasts demonstrated a 1-2 fold increase in (Na + + K + )-ATPase activity over the normal, while the transformed liver epithelial cells and carcinoma cells showed a 60% and 40% decrease in activity compared to the normal values, respectively. The 5'-nucleotidase activity was 2 to 3 times higher in the transformed fibroblasts

  1. Effects of ulipristal acetate on human embryo attachment and endometrial cell gene expression in an in vitro co-culture system.

    Science.gov (United States)

    Berger, C; Boggavarapu, N R; Menezes, J; Lalitkumar, P G L; Gemzell-Danielsson, K

    2015-04-01

    Does ulipristal acetate (UPA) used for emergency contraception (EC) interfere with the human embryo implantation process? UPA, at the dosage used for EC, does not affect human embryo implantation process, in vitro. A single pre-ovulatory dose of UPA (30 mg) acts by delaying or inhibiting ovulation and is recommended as first choice among emergency contraceptive pills due to its efficacy. The compound has also been demonstrated to have a dose-dependent effect on the endometrium, which theoretically could impair endometrial receptivity but its direct action on human embryo implantation has not yet been studied. Effect of UPA on embryo implantation process was studied in an in vitro endometrial construct. Human embryos were randomly added to the cultures and cultured for 5 more days with UPA (n = 10) or with vehicle alone (n = 10) to record the attachment of embryos. Endometrial biopsies were obtained from healthy, fertile women on cycle day LH+4 and stromal and epithelial cells were isolated. A three-dimensional in vitro endometrial co-culture system was constructed by mixing stromal cells with collagen covered with a layer of epithelial cells and cultured in progesterone containing medium until confluence. The treatment group received 200 ng/ml of UPA. Healthy, viable human embryos were placed on both control and treatment cultures. Five days later the cultures were tested for the attachment of embryos and the 3D endometrial constructs were analysed for endometrial receptivity markers by real-time PCR. There was no significant difference in the embryo attachment rate between the UPA treated group and the control group as 5 out of 10 human embryos exposed to UPA and 7 out of 10 embryos in the control group attached to the endometrial cell surface (P = 0.650). Out of 17 known receptivity genes studied here, only 2 genes, HBEGF (P = 0.009) and IL6 (P = 0.025) had a significant up-regulation and 4 genes, namely HAND2 (P = 0.003), OPN (P = 0.003), CALCR (P = 0.016) and

  2. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

    Science.gov (United States)

    Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.E.; Di Giulio, R.T.

    2001-01-01

    Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC50 and LD50 of TCDD for Fundulus embryos were ???19.7??9.5 pg TCDD/??l (water bath) and 0.25??0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction. Copyright ?? 2001 Elsevier Science B.V.

  3. Effects of embryo-derived exosomes on the development of bovine cloned embryos.

    Directory of Open Access Journals (Sweden)

    Pengxiang Qu

    Full Text Available The developmental competence of in vitro cultured (IVC embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE, as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development, but also following growth to term (in vivo development. Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.

  4. 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.

  5. Characterization of the onset of embryonic control and early development in the bovine embryo

    International Nuclear Information System (INIS)

    Barnes, F.L.

    1988-01-01

    Bovine embryos were used to determine if morphological and molecular features of early development are similar to in vivo recovered bovine embryos and to determine at what level early bovine development is regulated. Radiolabeling of IVP embryos and in vivo recovered embryos with 35 S-methionine for SDS-polyacrylamide gel electrophoresis reveals that these embryos are equivalent. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between late 8-cells and morulae. This transition is α-amanitin sensitive therefore due to de novo embryonic transcription. Embryonic transcription is partially responsible for terminating the post-transcriptionally regulated period of early bovine development. Argentophillic nucleolar organizing regions (Ag-NORs) indicate onset of nucleolar activation. Ag-NORs were absent in 2- and 4-cell IVP embryos and rarely occurred in 8-cell IVP embryos cultured in vitro. IVP 1- and 2-cell embryos cultured to blastocysts in sheep oviducts demonstrated Ag-NORs. Thus the lack of nucleolar activation of IVP embryos cultured in vitro is culture induced between the 2- and 8-cell stage

  6. 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.

  7. 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.

  8. 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.

  9. 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

  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. Label-free characterization of vitrification-induced morphology changes in single-cell embryos with full-field optical coherence tomography

    Science.gov (United States)

    Zarnescu, Livia; Leung, Michael C.; Abeyta, Michael; Sudkamp, Helge; Baer, Thomas; Behr, Barry; Ellerbee, Audrey K.

    2015-09-01

    Vitrification is an increasingly popular method of embryo cryopreservation that is used in assisted reproductive technology. Although vitrification has high post-thaw survival rates compared to other freezing techniques, its long-term effects on embryo development are still poorly understood. We demonstrate an application of full-field optical coherence tomography (FF-OCT) to visualize the effects of vitrification on live single-cell (2 pronuclear) mouse embryos without harmful labels. Using FF-OCT, we observed that vitrification causes a significant increase in the aggregation of structures within the embryo cytoplasm, consistent with reports in literature based on fluorescence techniques. We quantify the degree of aggregation with an objective metric, the cytoplasmic aggregation (CA) score, and observe a high degree of correlation between the CA scores of FF-OCT images of embryos and of fluorescence images of their mitochondria. Our results indicate that FF-OCT shows promise as a label-free assessment of the effects of vitrification on embryo mitochondria distribution. The CA score provides a quantitative metric to describe the degree to which embryos have been affected by vitrification and could aid clinicians in selecting embryos for transfer.

  12. Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

    DEFF Research Database (Denmark)

    Tao, Yong; Gheng, Lizi; Zhang, Meiling

    2008-01-01

    and dispered gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere......- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe...

  13. Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains.

    Science.gov (United States)

    Raymond, Gregory J; Raymond, Lynne D; Meade-White, Kimberly D; Hughson, Andrew G; Favara, Cynthia; Gardner, Donald; Williams, Elizabeth S; Miller, Michael W; Race, Richard E; Caughey, Byron

    2007-04-01

    In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.

  14. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

    International Nuclear Information System (INIS)

    Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

    1986-01-01

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells

  15. 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

  16. Caffeine induces metformin anticancer effect on fibrosarcoma in hamsters.

    Science.gov (United States)

    Popović, D J; Lalošević, D; Miljković, D; Popović, K J; Čapo, I; Popović, J K

    2018-04-01

    We investigated the effect of metformin and caffeine on fibrosarcoma in hamsters. 32 Syrian golden hamsters of both sexes, weighing approximately 100 g, were randomly allocated to 3 experimental and 2 control groups, with a minimum of 6 animals per group. 2 x 106 BHK-21/C13 cells in 1 ml were injected subcutaneously into the animals' back in 4 groups. The first experimental group started peroral treatment with metformin 500 mg/kg daily, the second with caffeine 100 mg/kg daily and the third with a combination of metformin 500 mg/kg and caffeine 100 mg/kg daily, via a gastric probe 3 days before tumor inoculation. After 2 weeks, when the tumors were approximately 2 cm in the control group, all animals were sacrificed. The blood was collected for glucose and other analyses. The tumors were excised and weighed and their diameters were measured. The tumor samples were pathohistologically (HE) and immunohistochemically (Ki-67, CD 31, COX IV, GLUT-1, iNOS) assessed and the main organs toxicologically analyzed, including the control animals that had received metformin and caffeine. Tumor volume was determined using the formula LxS2/2, where L was the longest and S the shortest diameter. Ki-67-positive cells in the tumor samples were quantified. Images were taken and processed by software UTHSCSA Image Tools for Windows Version 3.00. Statistical significances were determined by the Student's t-test. The combination of metformin and caffeine inhibited fibrosarcoma growth in hamsters without toxicity. Administration of metformin with caffeine might be an effective and safe approach in novel nontoxic adjuvant anticancer treatment.

  17. 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

  18. Developmental imaging: the avian embryo hatches to the challenge.

    Science.gov (United States)

    Kulesa, Paul M; McKinney, Mary C; McLennan, Rebecca

    2013-06-01

    The avian embryo provides a multifaceted model to study developmental mechanisms because of its accessibility to microsurgery, fluorescence cell labeling, in vivo imaging, and molecular manipulation. Early two-dimensional planar growth of the avian embryo mimics human development and provides unique access to complex cell migration patterns using light microscopy. Later developmental events continue to permit access to both light and other imaging modalities, making the avian embryo an excellent model for developmental imaging. For example, significant insights into cell and tissue behaviors within the primitive streak, craniofacial region, and cardiovascular and peripheral nervous systems have come from avian embryo studies. In this review, we provide an update to recent advances in embryo and tissue slice culture and imaging, fluorescence cell labeling, and gene profiling. We focus on how technical advances in the chick and quail provide a clearer understanding of how embryonic cell dynamics are beautifully choreographed in space and time to sculpt cells into functioning structures. We summarize how these technical advances help us to better understand basic developmental mechanisms that may lead to clinical research into human birth defects and tissue repair. Copyright © 2013 Wiley Periodicals, Inc.

  19. 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.

  20. 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...

  1. Induction of lyme arthritis in LSH hamsters

    International Nuclear Information System (INIS)

    Schmitz, J.L.; Schell, R.F.; Hejka, A.; England, D.M.; Konick, L.

    1988-01-01

    In studies of experimental Lyme disease, a major obstacle has been the unavailability of a suitable animal model. We found that irradiated LSH/Ss Lak hamsters developed arthritis after injection of Borrelia burgdorferi in the hind paws. When nonirradiated hamsters were injected in the hind paws with B. burgdorferi, acute transient synovitis was present. A diffuse neutrophilic infiltrate involved the synovia and periarticular structures. The inflammation was associated with edema, hyperemia, and granulation tissue. Numerous spirochetes were seen in the synovial and subsynovial tissues. The histopathologic changes were enhanced in irradiated hamsters. The onset and duration of the induced swelling were dependent on the dose of radiation and the inoculum of spirochetes. Inoculation of irradiated hamsters with Formalin-killed spirochetes or medium in which B. burgdorferi had grown for 7 days failed to induce swelling. This animal model should prove useful for studies of the immune response to B. burgdorferi and the pathogenesis of Lyme arthritis

  2. Chinese hamster ovary mutant UV-1 is hypomutable and defective in a postreplication recovery process

    International Nuclear Information System (INIS)

    Stamato, T.D.; Hinkle, L.; Collins, A.R.; Waldren, C.A.

    1981-01-01

    CHO-UV-1 is a mutant of the Chinese hamster cell CHO-K1 hypersensitive to killing by ultraviolet light but with normal resistance to X-ray. It is also hypersensitive to killing by ethyl methane sulfonate. Hybrid clones formed bu fusing UV-1 and Chinese hamster lung cells display the normal ultraviolet resistance of the latter. The sensitive phenotype behaves, therefore, in a genetically recessive manner. Ultraviolet sensitivity of UV-1 is not associated with a deficiency in excision repair. Alkaline sucrose gradient sedimentation analysis of nascent DNA from ultraviolet-irradiated cells reveals that UV-1 is, however, markedly deficient in postreplication recovery. Furthermore, UV-1 has a lower rate of induced mutation to 6-thioguanine resistance than does the parental cell when treated with ultraviolet light or ethyl methane sulfonate. These results suggest that the phenotype of UV-1 is due to a mutation in a form of postreplication recovery which in normal cells is error prone

  3. Autoradiographic demonstration of 3H-estradiol and 3H-cholesterol incorporation in hamster gonads

    International Nuclear Information System (INIS)

    Angelova, P.; Martinova, J.; Kyncheva, L.; Baleva-Ivanova, K.

    1989-01-01

    Male and female hamster gonads were investigated on day 14 of pregnancy, at birth, on days 7, 18 and 25 after birth and at sexual maturity. [2,4,6,7 3 H]-estradiol -17β, specific activity 110 Ci.mmol -1 and [1α, 2α - 3 H] - cholesterol specific activity 44 Ci.mmol -1 have been used for labelling. On embrional day 14 the histological image has been similar to that in the neonatal gonads - diffusive labelling includding germ, satellite and Leyding cells in fetal ovaries and testes. On the 7th postnatal day in the ovary a formation of primary follicles began in the deeper layers of gonads and an incorporation of the labelled substances in the germ and prefollicular cells in both ovary and testis have been observed. On the 18th postnatal day growing follicles have been seen in the ovary and labelling have been noticed in the oocytes and follicular cells. In the prepubertal testis the meiolic process has started, spermatocytes have been found and an incorporation of the radioactive substances in germ, Sertoli and Leydig cells has been established. In the ovaries of both 25th day old hamsters and adult animals multi-layered and preovulatory follicles have been seen. Sertoli cells, spermatogonia, spermatocytes and spertamids in the seminiferons tubules have been observed. The incorporation of 3 H-estradiol and 3 H cholesterol in both germ and Sertoli cells has been found. A presence has been observed of specific estradiol receptors in all three main cell types of fetal and developing gonads: germ, satellite and intertitial cells. The presence of estradiol receptors in developing hamster gonads has indicated a participation of steroids in the process of development and differentiation of male and female gonads

  4. Depletion of primordial germ cells (PGCs) by X-irradiation to extraembryonic region of chicken embryos and expression of xenotransplanted quail PGCs

    International Nuclear Information System (INIS)

    Atsumi, Y.; Yazawa, S.; Usui, F.; Nakamura, Y.; Yamamoto, Y.; Tagami, T.; Hiramatsu, K.; Kagami, H.; Ono, T.

    2009-01-01

    The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12 x 3 x 0.25 mm, - 0.1 g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120s (- 7.2 Gy) at stages 13 to 17 showed a hatchability of 35% (106/301), whereas the hatchability of unshielded embryos was 26% (27/105). The relative population of gonadal PGCs at stage 30 for embryos irradiated at stage 13 with or without shielding was 13 and 5%, respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or without shielding and transfused with quail embryonic blood containing PGCs each exhibited - 130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to - 7.2 Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras

  5. Fusion of blastomeres in mouse embryos under the action of femtosecond laser radiation. Efficiency of blastocyst formation and embryo development

    Energy Technology Data Exchange (ETDEWEB)

    Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A [N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow (Russian Federation); Ryabova, A V [A M Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation)

    2015-05-31

    Using the method of femtosecond laser surgery we study the fusion of two-cell mouse embryos under the action of tightly focused femtosecond laser radiation with the fusion efficiency reaching 60%. The detailed statistical analysis of the efficiency of blastomere fusion and development of the embryo up to the blastocyst stage after exposure of the embryos from different mice to a femtosecond pulse is presented. It is shown that the efficiency of blastocyst formation essentially depends on the biological characteristics of the embryo, namely, the strain and age of the donor mouse. The possibility of obtaining hexaploid embryonal cells using the methods of femtosecond laser surgery is demonstrated. (extreme light fields and their applications)

  6. 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.)

  7. 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)

  8. 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).

  9. Two-cell embryos are more sensitive than blastocysts to AMPK-dependent suppression of anabolism and stemness by commonly used fertility drugs, a diet supplement, and stress.

    Science.gov (United States)

    Bolnick, Alan; Abdulhasan, Mohammed; Kilburn, Brian; Xie, Yufen; Howard, Mindie; Andresen, Paul; Shamir, Alexandra M; Dai, Jing; Puscheck, Elizabeth E; Secor, Eric; Rappolee, Daniel A

    2017-12-01

    This study tests whether metformin or diet supplement BR-DIM-induced AMP-activated protein kinase (AMPK) mediated effects on development are more pronounced in blastocysts or 2-cell mouse embryos. Culture mouse zygotes to two-cell embryos and test effects after 0.5-1 h AMPK agonists' (e.g., Met, BR-DIM) exposure on AMPK-dependent ACCser79P phosphorylation and/or Oct4 by immunofluorescence. Culture morulae to blastocysts and test for increased ACCser79P, decreased Oct4 and for AMPK dependence by coculture with AMPK inhibitor compound C (CC). Test whether Met or BR-DIM decrease growth rates of morulae cultured to blastocyst by counting cells. Aspirin, metformin, and hyperosmotic sorbitol increased pACC ser79P ~ 20-fold, and BR-DIM caused a ~ 30-fold increase over two-cell embryos cultured for 1 h in KSOMaa but only 3- to 6-fold increase in blastocysts. We previously showed that these stimuli decreased Oct4 40-85% in two-cell embryos that was ~ 60-90% reversible by coculture with AMPK inhibitor CC. However, Oct4 decreased only 30-50% in blastocysts, although reversibility of loss by CC was similar at both embryo stages. Met and BR-DIM previously caused a near-complete cell proliferation arrest in two-cell embryos and here Met caused lower CC-reversible growth decrease and AMPK-independent BR-DIM-induced blastocyst growth decrease. Inducing drug or diet supplements decreased anabolism, growth, and stemness have a greater impact on AMPK-dependent processes in two-cell embryos compared to blastocysts.

  10. Development of porcine transgenic nuclear-transferred embryos derived from fibroblast cells transfected by the novel technique of nucleofection or standard lipofection.

    Science.gov (United States)

    Skrzyszowska, M; Samiec, M; Słomski, R; Lipiński, D; Mały, E

    2008-07-15

    The aim of our study was to determine the in vitro developmental potential of porcine nuclear-transferred (NT) embryos that had been reconstructed with Tg(pWAPhGH-GFPBsd) transgene-expressing fibroblast cells. The gene construct was introduced into fibroblast cells by the novel method of nucleofection or standard lipofection. NT oocytes derived from foetal and adult dermal fibroblast cells were stimulated by either simultaneous fusion and electrical activation (Groups IA and IB) or sequential electrical and chemical activation (Groups IIA and IIB). The percentages of cloned embryos that reached the morula and blastocyst stages were 152/254 (59.8%) and 77/254 (30.3%) or 139/276 (50.4%) and 45/276 (16.3%) in Groups IA or IB, respectively. The rates of NT embryos that developed to the morula and blastocyst stages were 103/179 (57.5%) and 41/179 (22.9%) or 84/193 (43.5%) and 27/193 (14.0%) in Groups IIA and IIB, respectively. In conclusion, the in vitro developmental competences of porcine transgenic NT embryos that had been reconstructed with the Tg(pWAPhGH-GFPBsd) gene-transfected fibroblast cells were relatively high. Further, the nucleofection efficiency of all the porcine fibroblast cell lines as estimated by intra-vitam fluorescent evaluation based on the index of reporter eGFP transgene expression was nearly 100%. However, PCR analysis for transgene screening confirmed the absence of Tg(pWAPhGH-GFPBsd) fusion gene in some of the nucleofected cell lines. To our knowledge, the novel method of nucleofection is the first to transfect nuclear donor cells in the production of transgenic cloned embryos.

  11. Cryopreservation of peach palm zygotic embryos.

    Science.gov (United States)

    Steinmacher, Douglas A; Saldanha, Cleber W; Clement, Charles R; Guerra, Miguel P

    2007-01-01

    Cryopreservation is a safe and cost-effective option for long-term germplasm conservation of non-orthodox seed species, such as peach palm (Bactris gasipaes). The objective of the present study was to establish a cryopreservation protocol for peach palm zygotic embryos based on the encapsulation-dehydration technique. After excision, zygotic embryos were encapsulated with 3 percent sodium alginate plus 2 M glycerol and 0.4 M sucrose, and pre-treated or not with 1 M sucrose during 24 h, followed by air-drying. Fresh weight water contents of beads decreased from 83 percent and 87 percent to 18 percent and 20 percent for pre-treated or non-pretreated beads, respectively, after 4 h of dehydration. Sucrose pre-treatment at 1 M caused lower zygotic embryo germination and plantlet height in contrast to non-treated beads. All the variables were statistically influenced by dehydration time. Optimal conditions for recovery of cryopreserved zygotic embryos include encapsulation and dehydration for 4 h in a forced air cabinet to 20 percent water content, followed by rapid freezing in liquid nitrogen (-196 degree C) and rapid thawing at 45 degree C. In these conditions 29 percent of the zygotic embryos germinated in vitro. However, plantlets obtained from dehydrated zygotic embryos had stunted haustoria and lower heights. Histological analysis showed that haustorium cells were large, vacuolated, with few protein bodies. In contrast, small cells with high nucleus:cytoplasm ratio formed the shoot apical meristem of the embryos, which were the cell types with favorable characteristics for survival after exposure to liquid nitrogen. Plantlets were successfully acclimatized and showed 41+/-9 percent and 88+/-4 percent survival levels after 12 weeks of acclimatization from cryopreserved and non-cryopreserved treatments, respectively.

  12. 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.)

  13. 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

  14. [TSA improve transgenic porcine cloned embryo development and transgene expression].

    Science.gov (United States)

    Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

    2011-07-01

    Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

  15. 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...

  16. 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)

  17. Role of microRNAs in embryo implantation

    Directory of Open Access Journals (Sweden)

    Jingjie Liang

    2017-11-01

    Full Text Available Abstract Failure of embryo implantation is a major limiting factor in early pregnancy and assisted reproduction. Determinants of implantation include the embryo viability, the endometrial receptivity, and embryo-maternal interactions. Multiple molecules are involved in the regulation of implantation, but their specific regulatory mechanisms remain unclear. MicroRNA (miRNA, functioning as the transcriptional regulator of gene expression, has been widely reported to be involved in embryo implantation. Recent studies reveal that miRNAs not only act inside the cells, but also can be released by cells into the extracellular environment through multiple packaging forms, facilitating intercellular communication and providing indicative information associated with physiological and pathological conditions. The discovery of extracellular miRNAs shed new light on implantation studies. MiRNAs provide new mechanisms for embryo-maternal communication. Moreover, they may serve as non-invasive biomarkers for embryo selection and assessment of endometrial receptivity in assisted reproduction, which improves the accuracy of evaluation while reducing the mechanical damage to the tissue. In this review, we discuss the involvement of miRNAs in embryo implantation from several aspects, focusing on the role of extracellular miRNAs and their potential applications in assisted reproductive technologies (ART to promote fertility efficiency.

  18. 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

  19. 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.

  20. 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.

  1. Laboratory techniques for human embryos.

    Science.gov (United States)

    Geber, Selmo; Sales, Liana; Sampaio, Marcos A C

    2002-01-01

    This review is concerned with laboratory techniques needed for assisted conception, particularly the handling of gametes and embryos. Such methods are being increasingly refined. Successive stages of fertilization and embryogenesis require especial care, and often involve the use of micromanipulative methods for intracytoplasmic sperm injection (ICSI) or preimplantation genetic diagnosis. Embryologists must take responsibility for gamete collection and preparation, and for deciding on the means of insemination or ICSI. Embryos must be assessed in culture, during the 1-cell, cleaving and morula/blastocyst stages, and classified according to quality. Co-culture methods may be necessary. The best embryos for transfer must be selected and loaded into the transfer catheter. Embryos not transferred must be cryopreserved, which demands the correct application of current methods of media preparation, seeding and the correct speed for cooling and warming. Before too long, methods of detecting abnormal embryos and avoiding their transfer may become widespread.

  2. Effects of alpha particles on zebrafish embryos

    International Nuclear Information System (INIS)

    Yum, E.H.W.; Choi, V.W.Y.; Yu, K.N.; Li, V.W.T.; Cheng, S.H.

    2008-01-01

    Full text: Ionizing radiation such as X-ray and alpha particles can damage cellular macromolecules, which can lead to DNA single- and double-strand breaks. In the present work, we studied the effects of alpha particles on dechorionated zebrafish embryos. Thin polyallyldiglycol carbonate (PADC) films with a thickness of 16 μm were prepared from commercially available PADC films (with thickness of 100 μm) by chemical etching and used as support substrates for holding zebrafish embryos for alpha-particle irradiation. These films recorded alpha-particle hit positions, quantified the number and energy of alpha particles actually incident on the embryo cells, and thus enabled the calculation of the dose absorbed by the embryo cells. Irradiation was made at 1.25 hours post fertilization (hpf) with various absorbed dose. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed on the embryos at different time stages after irradiation. Marked apoptosis was detected only in embryos at earlier time stages. The results showed that DNA double-strand break during zebrafish embryogenesis can be induced by alpha-particle irradiation, which suggests that zebrafish is a potential model for assessing the effects of alpha-particle radiation

  3. 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.

  4. Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

    Directory of Open Access Journals (Sweden)

    Yawei Gao

    2017-12-01

    Full Text Available Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst. We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

  5. 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)

  6. Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

    Directory of Open Access Journals (Sweden)

    Miguel A. Ibeas

    2017-12-01

    Full Text Available Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

  7. 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

  8. 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

  9. 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.

  10. 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)

  11. 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.

  12. 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.

  13. Cultures of preimplantation mouse embryos

    International Nuclear Information System (INIS)

    Streffer, C.; Molls, M.

    1987-01-01

    In the preimplantation mouse embryos the chromosomal damage develops through several postradiation cell cycles and mitoses. New chromosome aberrations are seen during the second and third postradiation mitoses. Also, more micronuclei appear during later postradiation interphases. This is in agreement with the assumption that unrepaired chromosomal radiation damage develops during the cell generation cycle to such a form (i.e. double-strand breaks in DNA) that chromosomal breaks occur. This proposition is strengthened by the observation that radiation-induced damage is more rapidly expressed after neutron exposure (first or second postradiation mitosis) than after exposure to X rays at the one- or two-cell stage. The preimplantation mouse embryo culture is an inviting system for additional studies at the molecular level, especially now that within the last few years more sensitive methods have been developed for study of DNA and protein structure, regulation, and synthesis. The results from these studies of cultures of preimplantation mouse embryos present a favorable case for the study of complex biological systems under very defined conditions in vitro for extrapolation to effects in vivo

  14. Analysis of structural and numerical chromosomal aberrations at the first and second mitosis after X irradiation of two-cell mouse embryos

    International Nuclear Information System (INIS)

    Weissenborn, U.; Streffer, C.

    1989-01-01

    Two-cell mouse embryos were X-irradiated in the late G2 phase in vivo. The first and second postradiation mitoses were analyzed for chromosomal anomalies. The majority of structural aberrations visible at the first mitosis after irradiation were chromatid breaks and chromatid gaps; only a few interchanges and dicentrics were observed. The aberration frequency resulted in a dose-effect relationship which was well described by a linear model. At the second mitosis 29% of the structural aberrations of the first mitosis were counted; the aberration quality changed only slightly. It is discussed whether these aberrations are to be considered new, derived, or unchanged transmitted aberrations. Contrary to the results obtained after irradiation of one-cell embryos, little chromosome loss was induced by radiation in two-cell embryos

  15. 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

  16. 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

  17. 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

  18. Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

    Science.gov (United States)

    Wong, Christopher Yee; Mills, James K

    2017-03-01

    Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

  19. Blood Vessel Normalization in the Hamster Oral Cancer Model for Experimental Cancer Therapy Studies

    Energy Technology Data Exchange (ETDEWEB)

    Ana J. Molinari; Romina F. Aromando; Maria E. Itoiz; Marcela A. Garabalino; Andrea Monti Hughes; Elisa M. Heber; Emiliano C. C. Pozzi; David W. Nigg; Veronica A. Trivillin; Amanda E. Schwint

    2012-07-01

    Normalization of tumor blood vessels improves drug and oxygen delivery to cancer cells. The aim of this study was to develop a technique to normalize blood vessels in the hamster cheek pouch model of oral cancer. Materials and Methods: Tumor-bearing hamsters were treated with thalidomide and were compared with controls. Results: Twenty eight hours after treatment with thalidomide, the blood vessels of premalignant tissue observable in vivo became narrower and less tortuous than those of controls; Evans Blue Dye extravasation in tumor was significantly reduced (indicating a reduction in aberrant tumor vascular hyperpermeability that compromises blood flow), and tumor blood vessel morphology in histological sections, labeled for Factor VIII, revealed a significant reduction in compressive forces. These findings indicated blood vessel normalization with a window of 48 h. Conclusion: The technique developed herein has rendered the hamster oral cancer model amenable to research, with the potential benefit of vascular normalization in head and neck cancer therapy.

  20. Active caspase-3 and ultrastructural evidence of apoptosis in spontaneous and induced cell death in bovine in vitro produced pre-implantation embryos

    DEFF Research Database (Denmark)

    Gjørret, Jakob O.; Fabian, Dusan; Avery, Birthe

    2007-01-01

    In this study we investigated chronological onset and involvement of active caspase-3, apoptotic nuclear morphology, and TUNEL-labeling, as well as ultrastructural evidence of apoptosis, in both spontaneous and induced cell death during pre-implantation development of bovine in vitro produced...... microscopy in both treated and untreated blastocysts. Activation of caspase-3 is likely involved in both spontaneous and induced apoptosis in bovine pre-implantation embryos, and immunohistochemical staining of active caspase-3 may be used in combination with other markers to identify apoptosis in pre...... embryos. Pre-implantation embryos (2-cell to Day 8 blastocysts) were cultured with either no supplementation (untreated) or with 10 µM staurosporine for 24 hr (treated). Embryos were subjected to immunohistochemical staining of active caspase-3, TUNEL-reaction for detection of DNA degradation and DAPI...

  1. 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.)

  2. 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

  3. 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)

  4. Treatment of Donor Cells and Reconstructed Embryos with a Combination of Trichostatin-A and 5-aza-2'-Deoxycytidine Improves the Developmental Competence and Quality of Buffalo Embryos Produced by Handmade Cloning and Alters Their Epigenetic Status and Gene Expression.

    Science.gov (United States)

    Saini, Monika; Selokar, Naresh L; Agrawal, Himanshu; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham S; Palta, Prabhat

    2017-06-01

    The application of cloning technology on a large scale is limited by very low offspring rate primarily due to aberrant or incomplete epigenetic reprogramming. Trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferases, are widely used for altering the epigenetic status of cloned embryos. We optimized the doses of these epigenetic modifiers for production of buffalo embryos by handmade cloning and examined whether combined treatment with these epigenetic modifiers offered any advantage over treatment with the individual epigenetic modifier. Irrespective of whether donor cells or reconstructed embryos or both were treated with 50 nM TSA +7.5 nM 5-aza-dC, (1) the blastocyst rate was significantly higher (71.6 ± 3.5, 68.3 ± 2.6, and 71.8 ± 2.4, respectively, vs. 43.1 ± 3.4 for controls, p cells or reconstructed embryos or both with the combination of TSA +5-aza-dC. Therefore, there is no advantage in treating both donor cells and reconstructed embryos when the combination of TSA and 5-aza-dC is used.

  5. 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)

  6. 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

  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. Molecular analysis of peroxisome proliferation in the hamster.

    Science.gov (United States)

    Choudhury, Agharul I; Sims, Helen M; Horley, Neill J; Roberts, Ruth A; Tomlinson, Simon R; Salter, Andrew M; Bruce, Mary; Shaw, P Nicholas; Kendall, David; Barrett, David A; Bell, David R

    2004-05-15

    Three novel P450 members of the cytochrome P450 4A family were cloned as partial cDNAs from hamster liver, characterised as novel members of the CYP4A subfamily, and designated CYP4A17, 18, and 19. Hamsters were treated with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, methylclofenapate (MCP) or Wy-14,643, and shown to develop hepatomegaly and induction of CYP4A17 RNA, and concomitant induction of lauric acid 12- hydroxylase. This treatment also resulted in hypolipidaemia, which was most pronounced in the VLDL fraction, with up to 50% reduction in VLDL-triglycerides; by contrast, blood cholesterol concentration was unaffected by this treatment. These data show that hamster is highly responsive to induction of CYP4A by peroxisome proliferators. To characterise the molecular basis of peroxisome proliferation, the hamster PPARalpha was cloned and shown to encode a 468-amino-acid protein, which is highly similar to rat and mouse PPARalpha proteins. The level of expression of hamster PPARalpha in liver is intermediate between mouse and guinea pig. These results fail to support the hypothesis that the level of PPARalpha in liver is directly responsible for species differences in peroxisome proliferation.

  9. Social context modulates food hoarding in Syrian hamsters

    Directory of Open Access Journals (Sweden)

    Bibiana Montoya

    2016-09-01

    Full Text Available The effect of the presence of a con-specific in the temporal organization of food hoarding was studied in two varieties of Syrian hamster (Mesocricetus auratus: golden and long-haired. Four male hamsters of each variety were used. Their foraging behavior was observed during four individual and four shared trials in which animals were not competing for the same food source or territory. During individual trials, long-haired hamsters consumed food items directly from the food source, transporting and hoarding only remaining pieces. During shared trials, the long-haired variety hoarded food items before consumption, and increased the duration of hoarding trips, food handling in the storage, and cache size. Golden hamsters maintained the same temporal organization of hoarding behavior (i.e., hoarding food items before consumption throughout both individual and shared trials. However, the golden variety increased handling time at the food source and decreased the duration of hoarding trips, the latency of hoarding and storing size throughout the shared trials. In Syrian hamsters, the presence of a con-specific may signal high probability of food source depletion suggesting that social pressures over food availability might facilitate hoarding behavior. Further studies are required to evaluate cost-benefit balance of food hoarding and the role of cache pilferage in this species.

  10. Gadolinium diethylenetriamine pentaacetic acid enhanced magnetic resonance imagings in cardiomyopathic hamsters. Histopathologic correlation

    International Nuclear Information System (INIS)

    Aso, Hiroko

    1995-01-01

    To assess the significance of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA)-enhanced magnetic resonance (MR) imaging, the findings were correlated with histopathological findings in cardiomyopathic hamsters (Bio 14.6). In hamsters given 1 mBq of Gd-DTPA, autoradiography revealed uptake of Gd-DTPA corresponding to the fibrotic tissue. According to the degree of fibrosis and inflammation, the tissue was graded into three. The ratio of contrast enhancement in the fibrotic area to that in the normal area was significantly higher in grade 1 than grades 2 and 3, and in grade 2 than grade 3. Next, hamsters in various age groups were given 0.2 mmol/kg intravenously. In the age group of 2-5 month, contrast enhancement was homogeneously observed in the entire myocardium. In the age group of 8-10 years, it was entirely observed, partly with heterogeneous enhancement. In the age group of 11-12 years, contrast enhancement was not different from that in the normal hamsters. Histological examination revealed that fibrosis changed from grade 1 through grade 3 with advancing age. In conclusion, MR imaging for myocardiopathy showed signal intensity reflecting the fibrotic tissue. Contrast enhancement of MR imaging was stronger when much more inflammatory cells were involved and fibrotic tissues were filled with much more blood vessels. Thus MR imaging may be a promising tool for evaluating the severity of myocardiopathy. (N.K.)

  11. Hamster and Murine Models of Severe Destructive Lyme Arthritis

    Science.gov (United States)

    Munson, Erik; Nardelli, Dean T.; Du Chateau, Brian K.; Callister, Steven M.; Schell, Ronald F.

    2012-01-01

    Arthritis is a frequent complication of infection in humans with Borrelia burgdorferi. Weeks to months following the onset of Lyme borreliosis, a histopathological reaction characteristic of synovitis including bone, joint, muscle, or tendon pain may occur. A subpopulation of patients may progress to a chronic, debilitating arthritis months to years after infection which has been classified as severe destructive Lyme arthritis. This arthritis involves focal bone erosion and destruction of articular cartilage. Hamsters and mice are animal models that have been utilized to study articular manifestations of Lyme borreliosis. Infection of immunocompetent LSH hamsters or C3H mice results in a transient synovitis. However, severe destructive Lyme arthritis can be induced by infecting irradiated hamsters or mice and immunocompetent Borrelia-vaccinated hamsters, mice, and interferon-gamma- (IFN-γ-) deficient mice with viable B. burgdorferi. The hamster model of severe destructive Lyme arthritis facilitates easy assessment of Lyme borreliosis vaccine preparations for deleterious effects while murine models of severe destructive Lyme arthritis allow for investigation of mechanisms of immunopathology. PMID:22461836

  12. Developmental and dysmorphogenic effects of glufosinate ammonium on mouse embryos in culture.

    Science.gov (United States)

    Watanabe, T; Iwase, T

    1996-01-01

    The effects of glufosinate ammonium on embryonic development in mice were examined using whole embryo and micromass cultures of midbrain and limb bud cells. In day 8 embryos cultured for 48 hr, glufosinate caused significant overall embryonic growth retardation and increased embryolethality to 37.5% at 10 micrograms/ml (5.0 x 10(-5) M). All embryos in the treated groups exhibited specific morphological defects including hypoplasia of the prosencephalon (forebrain) (100%) and visceral arches (100%). In day 10 embryos cultured for 24 hr, glufosinate significantly reduced the crown-rump length and the number of somite pairs, and produced a high incidence of morphological defects (84.6%) at 10 micrograms/ml. These embryos were characterized by blister in the lateral head (100%), hypoplasia of prosencephalon (57.1%), and cleft lips (42.9%) at 20 micrograms/ml (10.0 x 10(-5) M). Histological examination of the treated embryos showed numerous cell death (pyknotic debris) present throughout the neuroepithelium in the brain vesicle and neural tube, but did not involve the underlying mesenchyme. In micromass culture, glufosinate inhibited the differentiation of midbrain cells in day 12 embryos with 50% inhibition occurring at 0.55 microgram/ml (2.8 x 10(-6) M). The ratios of 50% inhibition concentration for cell proliferation to cell differentiation in limb bud cells were 0.76 and 1.52 in day 11 and 12 embryos, respectively. These findings indicate that glufosinate ammonium is embryotoxic in vitro. In addition to causing growth retardation, glufosinate specifically affected the neuroepithelium of the brain vesicle and neural tube, leading to neuroepithelial cell death.

  13. 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

  14. 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+

  15. Mouse one-cell embryos undergoing a radiation-induced G2 arrest may re-enter S-phase in the absence of cytokinesis

    International Nuclear Information System (INIS)

    Jacquet, P.; Buset, J.; Vankerkom, J.; Baatout, S.; De Saint-Georges, L.; Schoonjans, W.; Desaintes, C.

    2002-01-01

    PCC (premature chromosome condensation) can be used for visualizing and scoring damage induced by radiation in the chromatin of cells undergoing a G1 or G2 arrest. A method involving the fusion of irradiated single embryonic cells with single MI oocytes was used to induce PCC in mouse zygotes of the BALB/c strain, which suffer a drastic G2 arrest after X-irradiation (dose used 2.5 Gy). Other G2-arrested embryos were exposed in vitro to the phosphatase inhibitor calyculin A. Both methods furnished excellent chromosome preparations of the G2-arrested embryos. The mean number of chromosome fragments did not change significantly during G2 arrest, suggesting that zygotes of this strain are unable to repair DNA damage leading to such aberrations. Forty to fifty percent of the irradiated embryos were unable to cleave after G2 arrest and remained blocked at the one-cell stage for a few days before dying. PCC preparations obtained from such embryos suggested that about 30% of them had undergone a late mitosis not followed by cytokinesis and had entered a new DNA synthesis. These results are discussed in the light of recent observations in irradiated human cells deficient in the p53/14-3-3σ pathway. (author)

  16. 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.

  17. 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

  18. Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts

    International Nuclear Information System (INIS)

    Shaham, M.; Adler, B.; Ganguly, S.; Chaganti, R.S.K.

    1987-01-01

    Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). The authors report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental of Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 μgml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEB/sup r/) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEB/sup r/ colonies independently derived from transfection of human DNA and one of three DEB/sup r/ colonies independently derived from transfection of Chinese hamster DNA. The results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA

  19. cDNA cloning and nucleotide sequence comparison of Chinese hamster metallothionein I and II mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Griffith, B B; Walters, R A; Enger, M D; Hildebrand, C E; Griffith, J K

    1983-01-01

    Polyadenylated RNA was extracted from a cadmium resistant Chinese hamster (CHO) cell line, enriched for metal-induced, abundant RNA sequences and cloned as double-stranded cDNA in the plasmid pBR322. Two cDNA clones, pCHMT1 and pCHMT2, encoding two Chinese hamster isometallothioneins were identified, and the nucleotide sequence of each insert was determined. The two Chinese hamster metallothioneins show nucleotide sequence homologies of 80% in the protein coding region and approximately 35% in both the 5' and 3' untranslated regions. Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far. Estimated nucleotide substitution rates derived from interspecies comparisons were used to calculate a metallothionein gene duplication time of 45 to 120 million years ago. 39 references, 1 figure, 1 table.

  20. Correction of β-thalassemia mutant by base editor in human embryos

    Directory of Open Access Journals (Sweden)

    Puping Liang

    2017-09-01

    Full Text Available Abstract β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

  1. Effect of localized hypoxia on Drosophila embryo development.

    Directory of Open Access Journals (Sweden)

    Zhinan Wang

    Full Text Available Environmental stress, such as oxygen deprivation, affects various cellular activities and developmental processes. In this study, we directly investigated Drosophila embryo development in vivo while cultured on a microfluidic device, which imposed an oxygen gradient on the developing embryos. The designed microfluidic device enabled both temporal and spatial control of the local oxygen gradient applied to the live embryos. Time-lapse live cell imaging was used to monitor the morphology and cellular migration patterns as embryos were placed in various geometries relative to the oxygen gradient. Results show that pole cell movement and tail retraction during Drosophila embryogenesis are highly sensitive to oxygen concentrations. Through modeling, we also estimated the oxygen permeability across the Drosophila embryonic layers for the first time using parameters measured on our oxygen control device.

  2. Relation between radio-adaptive response and cell to cell communication

    International Nuclear Information System (INIS)

    Keiichiro Ishii

    1996-01-01

    Ionizing radiation has been considered to cause severe damages to DNA and do harm to cells in proportion to the dose, however low it might be. In 1984, Wolff et al. showed that human peripheral lymphocytes adapted to the low-dose radiation from 3 H-TdR added in culture medium and became resistant to the subsequent irradiation with high-doses of X-rays. This response, which is called radio-adaptive response, is also induced by X-rays and gamma-rays in human lymphocytes and Chinese hamster V79 cells. However, the mechanisms of and conditions for adaptive responses to radiation have not been clarified. With an objective of clarifying the conditions for adaptive responses of cells to radiation, we examined how the cell to cell communication is involved in the adaptive responses. We irradiated normal human embryo-derived (HE) cells and cancer cells (HeLa) in culture at high density with low-dose X-ray and examined their radio-adaptive responses by measuring the changes in sensitivity to subsequent high-dose X-ray irradiation using the Trypan Blue dye-exclusion test method. We also conducted experiments to examine the effects of Ca 2+ ions and Phorbol 12-Myristate 13-Acetate (TPA) which are supposed to be involved in cell to cell communication. (author)

  3. Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

    Science.gov (United States)

    Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

    2015-01-01

    Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

  4. Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

    Directory of Open Access Journals (Sweden)

    Sushil Kumar Mohapatra

    Full Text Available Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT has had a limited applicability due to very low (>1% live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF and Hand-made cloning (TE-HMC, and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

  5. Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

    Science.gov (United States)

    Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

    2015-01-01

    Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF. PMID:26053554

  6. Focus formation and neoplastic transformation by herpes simplex virus type 2 inactivated intracellularly by 5-bromo-2'-deoxyuridine and near UV light

    International Nuclear Information System (INIS)

    Manak, M.M.; Aurelian, L.; Ts'o, P.O.

    1981-01-01

    The induction of focus formation in low serum and of neoplastic transformation of Syrian hamster embryo cells was examined after the expression of herpes simplex virus type 2 functions. Syrian hamster embryo cells infected at a high multiplicity (5 PFU/cell) with 5-bromo-2'-deoxyuridine-labeled herpes simplex virus type 2 (11% substitution of thymidine residues) were exposed to near UV light irradiation at various times postinfection. This procedure specifically inactivated the viral genome, while having little, if any, effect on the unlabeled cellular DNA. Focus formation in 1% serum and neoplastic transformation were observed in cells exposed to virus inactivated before infection, but the frequency was enhanced (15- to 27-fold) in cells in which the virus was inactivated at 4 to 8 h postinfection. Only 2 to 45 independently isolated foci were capable of establishing tumorigenic lines. The established lines exhibited phenotypic alterations characteristic of a transformed state, including reduced serum requirement, anchorage-independent growth, and tumorigenicity. They retained viral DNA sequences and, even at relatively late passage, expressed viral antigens, including ICP 10

  7. Cystolithiasis in a Syrian hamster: a different outcome | Petrini ...

    African Journals Online (AJOL)

    Considering the positive outcome and the beneficial properties of palmitoylethanolamide, glucosamine, and hesperidin, these nutritional elements in Syrian hamsters, are recommended to reduce recurrence after surgical treatment of urolithiasis. Keywords: Glucosamine, Hamster, Hesperidin, PEA, Urolithiasis ...

  8. Analysis of metastasis of melanoma-bearing hamsters in thermal neutron capture therapy

    International Nuclear Information System (INIS)

    Ueda, Masataka; Mishima, Yutaka; Ichihashi, Masamitsu

    1985-01-01

    Melanoma-bearing hamsters were divided into three groups: the MG I was treated with both 10 B 1 -para-boronophenylalanine.HCl ( 10 B 1 -BPA) and neutron capture therapy (NCT); the MG II was treated with NCT alone; and the control group (MG III). The most satisfactory effect on regression was seen in the MG I. When the opposite site to the transplanted tumor site was exposed to thermal neutrons, no enhanced effect on metastasis was seen. Tumor cells of MG I and MG II were transplanted subcutaneously 24 hr after NCT into normal hamsters (MG It and MG IIt), and their growth and metastasis abilities were examined. MG It cells possessed neither growth nor metastasis ability; while MG IIt cells showed normal growth and metastasis abilities. Lethal effects on tumor cells seemed to occur in the MG I at 24 hr after NCT, suggesting no effects of NCT on the metastasis ability of tumor cells. Metastasis was seen in 2 of 8 animals in the MG III; however, inhibitory effects on tumor cells were the same as those in the other groups MG I and MG II. When the cells were exposed to 100 rad and 300 rad of gamma rays to assess effects of gamma rays during NCT, neither tumor growth nor lung metastasis was affected. When the tumor was excised with 5 mm margin, relapse occurred in a high incidence. There was no difference in lung metastasis between NCT and gamma irradiation. (Namekawa, K.)

  9. 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.

  10. Inhibitory effects of Zengshengping fractions on DMBA-induced buccal pouch carcinogenesis in hamsters.

    Science.gov (United States)

    Guan, Xiao-Bing; Sun, Zheng; Chen, Xiao-Xin; Wu, Hong-Ru; Zhang, Xin-Yan

    2012-01-01

    Zengshengping (ZSP) tablets had inhibitory effects on oral precancerous lesions by reducing the incidence of oral cancer. However, the severe liver toxicity caused by systemic administration of ZSP limits the long-term use of this anti-cancer drug. The purpose of this study was to evaluate the tumor inhibitory effects due to the topical application of extracts from ZSP, a Chinese herbal drug, on 7, 12-dimethlbenz(a)anthracene (DMBA) induced oral tumors in hamsters. The study also investigated the anti-cancer mechanisms of the ZSP extracts on oral carcinogenesis. DMBA (0.5%) was applied topically to the buccal pouches of Syrian golden hamsters (6 - 8 weeks old) three times per week for six weeks in order to induce the development of oral tumors. Different fractions of ZSP were either applied topically to the oral tumor lesions or fed orally at varying dosages to animals with oral tumors for 18 weeks. Tumor volume was measured by histopathological examination. Tumor cell proliferation was evaluated by counting BrdU labeled cells and by Western blotting for mitogen-activated protein kinase (MAPK) protein levels. The protein levels of apoptosis marker Caspase-3 and regulator Bcl-2 protein were also measured by Western blotting. Topical application of DMBA to the left pouch of hamsters induced oral tumor formation. Animals treated with DMBA showed a loss in body weight while animals treated with ZSP maintained normal body weights. Both the ZSP n-butanol fraction and water fraction significantly reduced tumor volume by 32.6% (P oral tumor lesions and reduced the expression level of MAPK. In addition, ZSP promoted tumor cell apoptosis by increasing Caspase-3 expression but decreasing Bcl-2 protein production. The n-butanol and water fractions of ZSP are effective at inhibiting tumor cell proliferation and stimulating apoptosis in oral cancer suggesting that these fractions have chemopreventive effects on DMBA induced oral carcinogenesis.

  11. 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)

  12. Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae).

    Science.gov (United States)

    González-Gutiérrez, Alejandra G; Gutiérrez-Mora, Antonia; Rodríguez-Garay, Benjamín

    2014-01-01

    Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer's-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.

  13. [The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

    Science.gov (United States)

    Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

    1995-01-01

    A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

  14. 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.)

  15. Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification.

    Science.gov (United States)

    Eguiara, Arrate; Holgado, Olaia; Beloqui, Izaskun; Abalde, Leire; Sanchez, Yolanda; Callol, Carles; Martin, Angel G

    2011-11-01

    The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.

  16. Autoradiographic demonstration of sup 3 H-estradiol and sup 3 H-cholesterol incorporation in hamster gonads

    Energy Technology Data Exchange (ETDEWEB)

    Angelova, P; Martinova, J; Kyncheva, L; Baleva-Ivanova, K [Bylgarska Akademiya na Naukite, Sofia (Bulgaria). Inst. po Morfologiya

    1989-01-01

    Male and female hamster gonads were investigated on day 14 of pregnancy, at birth, on days 7, 18 and 25 after birth and at sexual maturity. (2,4,6,7 {sup 3}H)-estradiol -17{beta}, specific activity 110 Ci.mmol{sup -1} and (1{alpha}, 2{alpha} -{sup 3}H) - cholesterol specific activity 44 Ci.mmol{sup -1} have been used for labelling. On embrional day 14 the histological image has been similar to that in the neonatal gonads - diffusive labelling includding germ, satellite and Leyding cells in fetal ovaries and testes. On the 7th postnatal day in the ovary a formation of primary follicles began in the deeper layers of gonads and an incorporation of the labelled substances in the germ and prefollicular cells in both ovary and testis have been observed. On the 18th postnatal day growing follicles have been seen in the ovary and labelling have been noticed in the oocytes and follicular cells. In the prepubertal testis the meiolic process has started, spermatocytes have been found and an incorporation of the radioactive substances in germ, Sertoli and Leydig cells has been established. In the ovaries of both 25th day old hamsters and adult animals multi-layered and preovulatory follicles have been seen. Sertoli cells, spermatogonia, spermatocytes and spertamids in the seminiferons tubules have been observed. The incorporation of {sup 3}H-estradiol and {sup 3}H cholesterol in both germ and Sertoli cells has been found. A presence has been observed of specific estradiol receptors in all three main cell types of fetal and developing gonads: germ, satellite and intertitial cells. The presence of estradiol receptors in developing hamster gonads has indicated a participation of steroids in the process of development and differentiation of male and female gonads.

  17. Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

    Science.gov (United States)

    Shi, Li-Hong; Miao, Yi-Liang; Ouyang, Ying-Chun; Huang, Jun-Cheng; Lei, Zi-Li; Yang, Ji-Wen; Han, Zhi-Ming; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

    2008-03-01

    The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. (c) 2008 Wiley-Liss, Inc.

  18. Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice.

    Science.gov (United States)

    Binder, N K; Evans, J; Gardner, D K; Salamonsen, L A; Hannan, N J

    2014-10-10

    Does vascular endothelial growth factor (VEGF) have important roles during early embryo development and implantation? VEGF plays key roles during mouse preimplantation embryo development, with beneficial effects on time to cavitation, blastocyst cell number and outgrowth, as well as implantation rate and fetal limb development. Embryo implantation requires synchronized dialog between maternal cells and those of the conceptus. Following ovulation, secretions from endometrial glands increase and accumulate in the uterine lumen. These secretions contain important mediators that support the conceptus during the peri-implantation phase. Previously, we demonstrated a significant reduction of VEGFA in the uterine cavity of women with unexplained infertility. Functional studies demonstrated that VEGF significantly enhanced endometrial epithelial cell adhesive properties and embryo outgrowth. Human endometrial lavages (n = 6) were obtained from women of proven fertility. Four-week old Swiss mice were superovulated and mated with Swiss males to obtain embryos for treatment with VEGF in vitro. Preimplantation embryo development was assessed prior to embryo transfer (n = 19-30/treatment group/output). Recipient F1 female mice (8-12 weeks of age) were mated with vasectomized males to induce pseudopregnancy and embryos were transferred. On Day 14.5 of pregnancy, uterine horns were collected for analysis of implantation rates as well as placental and fetal development (n = 14-19/treatment). Lavage fluid was assessed by western immunoblot analysis to determine the VEGF isoforms present. Mouse embryos were treated with either recombinant human (rh)VEGF, or VEGF isoforms 121 and 165. Preimplantation embryo development was quantified using time-lapse microscopy. Blastocysts were (i) stained for cell number, (ii) transferred to wells coated with fibronectin to examine trophoblast outgrowth or (iii) transferred to pseudo pregnant recipients to analyze implantation rates, placental and

  19. 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.

  20. 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...

  1. Species difference between rat and hamster in tissue accumulation of mercury after administration of methylmercury

    International Nuclear Information System (INIS)

    Omata, Saburo; Kasama, Hidetaka; Hasegawa, Hiroshi; Hasegawa, Kazuhiro; Sugano, Hiroshi; Ozaki, Kunio

    1986-01-01

    The accumulation of mercury in tissues of the rat and hamster was determined after the administration of a single dose of 203 Hg-methylmercury chloride (10 mg/kg body weight). (1) On day 2, the mercury contents of hamster tissues were higher than those of rat tissues, except for red blood cells, in which the mercury content was about 6-fold higher in the rat than in the hamster. (2) After that time, the mercury content of hamster tissues decreased rather steeply and on day 16 it had reached 14-25% in nervous tissues and 7-15% in other tissues, of the levels on day 2. (3) In the rat, on the other hand, the mercury content of nervous tissues on day 16 was higher than that on day 2 (106-220%), except for dorsal roots and dorsal root ganglia, which showed slight decreases (75-94% of the levels on day 2). In non-neural tissues, the decreases up to day 16 were also small (71-92% of the levels on day 2). (4) Thus, both the uptake and elimination of mercury seem to be more rapid in the tissues of hamster compared with those of the rat. Similar trends of mercury accumulation and elimination were observed when animals received multiple injections of methylmercury that induced acute methylmercury intoxication. (5) Significant biotransmormation of the injected methylmercury to inorganic mercury was detected in the liver, kidney and spleen of both animal species. Although the percentages of inorganic mercury in these tissues wer not so different between the two species on day 2, they became exceedingly high in the tissues of hamster at the later stage, except in the kidney cytosol, in which the values were close in both animal species between day 2 and day 16. (orig.)

  2. Subcellular distribution of Pu-239 in the liver of rat, mouse, Syrian and Chinese hamster

    International Nuclear Information System (INIS)

    Winter, R.; Seidel, A.

    1980-01-01

    The aim of our studies was to elucidate the biochemical mechanisms responsible for the differences in the biological half life of actinides in the liver of different mammalian species. Rats and mice were chosen as models for rapid elimination, and Syrian and Chinese hamsters as models for slow elimination. To distinguish between fixation in lysosomes and mitochondria, the lysosomes were isolated following injection of Triton WR1339 6 days after 239 Pu administration. The animals were sacrificed 4 days later. In order to study the possible association with ferritin, 59 Fe was also injected. Liver homogenates were subjected to differential and isopycnic centrifugation in a sucrose density gradient. The typical shift in the density of the lysosomal marker acid phosphatase from rho approximately 1.2 to rho approximately 1.1 following Triton WR1339 injection was observed in all species. It was possible therefore to separate lysosomes from other cell organelles, especially mitochondria. It was concluded that: 1) Mitochondria can virtually be excluded as binding sites in all four species; 2) Lysosomes are one important storage site in rats, mice and Syrian hamsters; 3) If 239 Pu is bound to another cell constituent in addition to lysosomes in the hamster species (which is not yet proven) its density should be approximately 1.17. (H.K.)

  3. 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

  4. Influence of the radiation (Co60) in pre-implants rabbit embryos: effect on atypic mitotic index and embryo pole development

    International Nuclear Information System (INIS)

    Approbato, Mario S.; Oliveira Moura, Katia K.V. de; Souza Florencio, Rodopiano de; Garcia, Ricardo; Faria, Renato S.; Benedetti, Leonardo N.; Goulart, Flamarion B.

    1995-01-01

    We studied the effect of ionizing irradiation on 12 New Zealand rabbits (65 embryos), at three different times: at match time (zero hour), two days after and four days after, with two different irradiation doses: five c Gy and ten c Gy. Six rabbits (36 blastocysts) were used as controls. the matching instant was the zero hour. Exactly six days after (± 60 minutes) the embryos of each rabbit was picked up by flushing the uterus with culture media. the embryos were fixed in methanol for 48 hours, and colored with acid Mayer hematoxylin. The following embryo parameters were studied: embryo pole development; percentage of abnormal mitotic figures. irradiation time was associated with lower scores of embryo pole development, but not with irradiation dose. There were no gross abnormalities of embryo pole. The abnormal mitotic cells was affected both by the time and dose of irradiation. (author)

  5. 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...

  6. HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

    Science.gov (United States)

    Levesque-Tremblay, Gabriel; Müller, Kerstin; Mansfield, Shawn D; Haughn, George W

    2015-03-01

    Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy. © 2015 American Society of Plant Biologists. All Rights Reserved.

  7. Evaluation of cell number and DNA content in mouse embryos cultivated with uranium; Evaluacion del numero de celulas y el contenido de DNA en embriones murinos cultivados con uranio

    Energy Technology Data Exchange (ETDEWEB)

    Kundt, Mirian S; Cabrini, Romulo L [Comision Nacional de Energia Atomica, General San Martin (Argentina). Dept. de Radiobiologia

    2000-07-01

    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 {mu}gU/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

  8. 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

  9. Protein synthesis in the embryo of Pinus thunbergii seed, 2

    International Nuclear Information System (INIS)

    Yamamoto, Naoaki; Sasaki, Satohiko.

    1977-01-01

    14 C-Amino acid incorporating activity in the absence of exogenous mRNA was found in a cell-free system from embryos of light-germinated Pinus thunbergii seeds, but not in that from dark-imbibed seed embryos. Template activity in the cell-free system from the light-germinated seed embryos was observed in the ribosome fraction, especially the polyribosome fraction, but not in the 100,000 x g supernatant fraction (s100). These facts suggest that the nature of the block in protein synthesis during the imbibition of seeds in the dark is due to the lack or inactivity of mRNA. The s100 from light-germinated seed embryos was found to be less active in amino acid incorporation than that from dark-imbibed seed embryos. (auth.)

  10. Histopathology of Lyme arthritis in LSH hamsters

    International Nuclear Information System (INIS)

    Hejka, A.; Schmitz, J.L.; England, D.M.; Callister, S.M.; Schell, R.F.

    1989-01-01

    The authors studied the histopathologic evolution of arthritis in nonirradiated and irradiated hamsters infected with Borrelia burgdorferi. Nonirradiated hamsters injected in the hind paws with B. burgdorferi developed an acute inflammatory reaction involving the synovium, periarticular soft tissues, and dermis. This acute inflammatory reaction was short-lived and was replaced by a mild chronic synovitis as the number of detectable spirochetes in the synovium, periarticular soft tissues, and perineurovascular areas diminished. Exposing hamsters to radiation before inoculation with B. burgdorferi exacerbated and prolonged the acute inflammatory phase. Spirochetes also persisted longer in the periarticular soft tissues. A major histopathologic finding was destructive and erosive bone changes of the hind paws, which resulted in deformation of the joints. These studies should be helpful in defining the immune mechanism participating in the onset, progression, and resolution of Lyme arthritis

  11. Regulation of membrane fusion and secretory events in the sea urchin embryo

    International Nuclear Information System (INIS)

    Roe, J.L.

    1990-01-01

    Membrane fusion and secretory events play a key role in fertilization and early development in the sea urchin embryo. To investigate the mechanism of membrane fusion, the effect of inhibitors of metalloendoprotease activity was studied on two model systems of cell fusion; fertilization and spiculogenesis by primary mesenchyme cells in the embryo. Both the zinc chelator, 1,10-phenanthroline, and peptide metalloprotease substrates were found to inhibit both fertilization and gamete fusion, while peptides that are not substrates of metalloproteases did not affect either process. Primary mesenchyme cells form the larval skeleton in the embryo by deposition of mineral and an organic matrix into a syncytial cavity formed by fusion of filopodia of these cells. Metalloprotease inhibitors were found to inhibit spiculogenesis both in vivo and in cultures of isolated primary mesenchyme cells, and the activity of a metalloprotease of the appropriate specificity was found in the primary mesenchyme cells. These two studies implicate the activity of a metalloprotease in a necessary step in membrane fusion. Following fertilization, exocytosis of the cortical granules results in the formation of the fertilization envelope and the hyaline layer, that surround the developing embryo. The hatching enzyme is secreted by the blastula stage sea urchin embryo, which proteolyzes the fertilization envelope surrounding the embryo, allowing the embryo to hatch. Using an assay that measures 125 I-fertilization envelope degradation, the hatching enzyme was identified as a 33 kDa metalloprotease, and was purified by ion-exchange and affinity chromatography from the hatching media of Strongylocentrotus purpuratus embryos. The hatching enzyme showed a substrate preference for only a minor subset of fertilization envelope proteins

  12. Photoperiodic regulation of the hamster testis: dependence on circadian rhythms

    International Nuclear Information System (INIS)

    Eskes, G.A.; Zucker, I.

    1978-01-01

    The testes of hamsters exposed to short days (10 hr of light per day) regress within 13 weeks. Administration of 7.5 percent deuterium oxide to hamsters lengthens the period of free running circadian activity rhythms by 2.2 percent and prevents testicular regression during short-day exposure. This is consistent with predictions derived from an external coincidence model for photoperiodic time measurement: Deuterium oxide changes phase relationships between the light-dark cycle and the circadian system, the hamster's daily photosensitive phase is stimulated with light during short days, and the testes remain large. Conservation of the period of circadian rhythms within narrow limits has adaptive significance for hamster photoperiodism and for the occurrence and phasing of the annual reproductive cycle

  13. Effect of embryo density on in vitro developmental characteristics of bovine preimplantative embryos with respect to micro and macroenvironments.

    Science.gov (United States)

    Hoelker, M; Rings, F; Lund, Q; Phatsara, C; Schellander, K; Tesfaye, D

    2010-10-01

    To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r(2) = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose. © 2009 Blackwell Verlag GmbH.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Effect of Radiofrequency Radiation Emitted from 2G and 3G Cell Phone on Developing Liver of Chick Embryo - A Comparative Study.

    Science.gov (United States)

    D'Silva, Mary Hydrina; Swer, Rijied Thompson; Anbalagan, J; Rajesh, Bhargavan

    2017-07-01

    The increasing scientific evidence of various health hazards on exposure of Radiofrequency Radiation (RFR) emitted from both the cell phones and base stations have caused significant media attention and public discussion in recent years. The mechanism of interaction of RF fields with developing tissues of children and fetuses may be different from that of adults due to their smaller physical size and variation in tissue electromagnetic properties. The present study may provide an insight into the basic mechanisms by which RF fields interact with developing tissues in an embryo. To evaluate the possible tissue and DNA damage in developing liver of chick embryo following chronic exposure to Ultra-High Frequency/Radiofrequency Radiation (UHF/RFR) emitted from 2G and 3G cell phone. Fertilized chick embryos were incubated in four groups. Group A-experimental group exposed to 2G radiation (60 eggs), Group B- experimental group exposed to 3G radiation (60 eggs), Group C- sham exposed control group (60 eggs) and Group D- control group (48 eggs). On completion of scheduled duration, the embryos were collected and processed for routine histological studies to check structural changes in liver. The nuclear diameter and karyorrhexis changes of hepatocytes were analysed using oculometer and square reticule respectively. The liver procured from one batch of eggs from all the four groups was subjected to alkaline comet assay technique to assess DNA damage. The results were compared using one-way ANOVA test. In our study, the exposure of developing chick embryos to 2G and 3G cell phone radiations caused structural changes in liver in the form of dilated sinusoidal spaces with haemorrhage, increased vacuolations in cytoplasm, increased nuclear diameter and karyorrhexis and significantly increased DNA damage. The chronic exposure of chick embryo liver to RFR emitted from 2G and 3G cell phone resulted in various structural changes and DNA damage. The changes were more pronounced in 3

  20. A cutin fluorescence pattern in developing embryos of some angiosperms

    Directory of Open Access Journals (Sweden)

    Ewa Szczuka

    2014-01-01

    Full Text Available A cuticle visualized by auramine O fluorescence appears on the developing embryos of 9 species belonging to Cruciferae, Caryophyllaceae, Plantaginaceae, Linaceae and Papilionaceae. In the investigated species the formation and extent of fluorescing and non-fluorescing embryonic areas follow a similar pattern. At first the cutin fluorescing layer is formed on the apical part of the proembryo without delimited protoderm. This layer extends and at the late globular stage envelops the embryo proper, except for a cell adjoining the suspensor. Fluorescing cutin persists during the heart stage but disappears from the torpedo embryo. During these stages there is no cutine fluorescence on suspensorial cells. Continuous cutin fluorescence appears again on the surface of the whole embryo by the late torpedo stage. Then fluorescence disappears from the radicular part of U-shaped embryos, but persists on the shoot apex, cotyledons and at least on the upper part of hypocotyl. It is assumed that polarization and nutrition of the embryo may be influenced by cuticular changes.

  1. 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...

  2. Raman Spectroscopic Imaging of the Whole Ciona intestinalis Embryo during Development

    Science.gov (United States)

    Nakamura, Mitsuru J.; Hotta, Kohji; Oka, Kotaro

    2013-01-01

    Intracellular composition and the distribution of bio-molecules play central roles in the specification of cell fates and morphogenesis during embryogenesis. Consequently, investigation of changes in the expression and distribution of bio-molecules, especially mRNAs and proteins, is an important challenge in developmental biology. Raman spectroscopic imaging, a non-invasive and label-free technique, allows simultaneous imaging of the intracellular composition and distribution of multiple bio-molecules. In this study, we explored the application of Raman spectroscopic imaging in the whole Ciona intestinalis embryo during development. Analysis of Raman spectra scattered from C. intestinalis embryos revealed a number of localized patterns of high Raman intensity within the embryo. Based on the observed distribution of bio-molecules, we succeeded in identifying the location and structure of differentiated muscle and endoderm within the whole embryo, up to the tailbud stage, in a label-free manner. Furthermore, during cell differentiation, we detected significant differences in cell state between muscle/endoderm daughter cells and daughter cells with other fates that had divided from the same mother cells; this was achieved by focusing on the Raman intensity of single Raman bands at 1002 or 1526 cm−1, respectively. This study reports the first application of Raman spectroscopic imaging to the study of identifying and characterizing differentiating tissues in a whole chordate embryo. Our results suggest that Raman spectroscopic imaging is a feasible label-free technique for investigating the developmental process of the whole embryo of C. intestinalis. PMID:23977129

  3. Supplementary heat-killed Lactobacillus reuteri GMNL-263 ameliorates hyperlipidaemic and cardiac apoptosis in high-fat diet-fed hamsters to maintain cardiovascular function.

    Science.gov (United States)

    Ting, Wei-Jen; Kuo, Wei-Wen; Kuo, Chia-Hua; Yeh, Yu-Lan; Shen, Chia-Yao; Chen, Ya-Hui; Ho, Tsung-Jung; Viswanadha, Vijaya Padma; Chen, Yi-Hsing; Huang, Chih-Yang

    2015-09-14

    Obesity and hyperlipidaemia increase the risk of CVD. Some strains of probiotics have been suggested to have potential applications in cardiovascular health by lowering serum LDL-cholesterol. In this work, high-fat diet-induced hyperlipidaemia in hamsters was treated with different doses (5×108 and 2·5×109 cells/kg per d) of heat-killed Lactobacillus reuteri GMNL-263 (Lr263) by oral gavage for 8 weeks. The serum lipid profile analysis showed that LDL-cholesterol and plasma malondialdehyde (P-MDA) were reduced in the GMNL-263 5×108 cells/kg per d treatment group. Total cholesterol and P-MDA were reduced in the GMNL-263 2·5×109 cells/kg per d treatment group. In terms of heart function, the GMNL-263 2·5×109 cells/kg per d treatments improved the ejection fraction from 85·71 to 91·81 % and fractional shortening from 46·93 to 57·92 % in the high-fat diet-fed hamster hearts. Moreover, the GMNL-263-treated, high-fat diet-fed hamster hearts exhibited reduced Fas-induced myocardial apoptosis and a reactivated IGF1R/PI3K/Akt cell survival pathway. Interestingly, the GMNL-263 treatments also enhanced the heat-shock protein 27 expression in a dose-dependent manner, but the mechanism for this increase remains unclear. In conclusion, supplementary heat-killed L. reuteri GMNL-263 can slightly reduce serum cholesterol. The anti-hyperlipidaemia effects of GMNL-263 may reactivate the IGF1R/PI3K/Akt cell survival pathway and reduce Fas-induced myocardial apoptosis in high-fat diet-fed hamster hearts.

  4. Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species.

    Science.gov (United States)

    Lagutina, Irina; Zakhartchenko, Valeri; Fulka, Helena; Colleoni, Silvia; Wolf, Eckhard; Fulka, Josef; Lazzari, Giovanna; Galli, Cesare

    2011-04-01

    The most successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos has been achieved in closely related species. The analyses of embryonic gene activity in iSCNT embryos of different species combinations have revealed the existence of significant aberrations in expression of housekeeping genes and genes dependent on the major embryonic genome activation (EGA). However, there are many studies with successful blastocyst (BL) development of iSCNT embryos derived from donor cells and oocytes of animal species with distant taxonomical relations (inter-family/inter-class) that should indicate proper EGA at least in terms of RNA polymerase I activation, nucleoli formation, and activation of genes engaged in morula and BL formation. We investigated the ability of bovine, porcine, and rabbit oocytes to activate embryonic nucleoli formation in the nuclei of somatic cells of different mammalian species. In iSCNT embryos, nucleoli precursor bodies originate from the oocyte, while most proteins engaged in the formation of mature nucleoli should be transcribed from genes de novo in the donor nucleus at the time of EGA. Thus, the success of nucleoli formation depends on species compatibility of many components of this complex process. We demonstrate that the time and cell stage of nucleoli formation are under the control of recipient ooplasm. Oocytes of the studied species possess different abilities to support nucleoli formation. Formation of nucleoli, which is a complex but small part of the whole process of EGA, is essential but not absolutely sufficient for the development of iSCNT embryos to the morula and BL stages.

  5. Human embryo cloning prohibited in Hong Kong.

    Science.gov (United States)

    Liu, Athena

    2005-12-01

    Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

  6. 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....

  7. Studies on the cytodifferentiation of the neuroblasts and visual cells in the chick embryo retina, using the electron-microscopic autoradiography of 3H-thymidine

    International Nuclear Information System (INIS)

    Mishima, H.; Fujita, H.

    1978-01-01

    Studies on the histogenetic analysis of cytodifferentiation of the neuroblast and visual cell in the chick embryo retina were made using the autoradiography of 3 H-thymidine. The posterior pole region of the eyeball was observed in all the animals used. The retina in a 4-day-old chick embryo consists exclusively of matrix cells forming the matrix layer. In a 5-day-old chick embryo retina, neuroblasts first differentiated from the matrix cells migrate into the outer part of the matrix layer, forming the mantle layer. The matrix cell is a homogeneous epithelial cell containing abundant free ribosomes and a poorly developed cytoplasmic membrane system in the cytoplasm. The characteristic sign of differentiation of the neuroblast is an appearance of elements of rough-surfaced endoplasmic reticulum and an indentation of the nucleus. The primitive visual cell having just lost its ability to synthesize DNA appears just beneath the pigment epithelium in a 7-day-old chick embryo, and all the cells lying beneath the pigment epithelium lose the ability to synthesize DNA at 10 days of incubation. The cytoplasmic process of the matrix cell is in contact with the adjacent one, making an apicolateral junction. When the matrix cell loses its ability to synthesize DNA, a big tentlike process extending over the level of the apicolateral junction appears. This phenomenon is considered to be a sign of differentiation from matrix cell to primitive visual cell, and this big tentlike process containing 2 centrioles is a primordium of the inner segment of the visual cell. (orig.) [de

  8. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

    Science.gov (United States)

    Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

    2013-10-01

    Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The

  9. 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...

  10. 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.

  11. Study of embryonic ploidy: a probable embryo model

    Energy Technology Data Exchange (ETDEWEB)

    Kundt, Miriam S; Cabrini, Romulo L [Comision Nacional de Energia Atomica, Buenos Aires (Argentina). Dept. de Radiobiologia

    2001-07-01

    The second polar body (PB) studies in preimplantation mouse embryos were carried out to evaluate the possibility as reference cell to analyze ploidy. For that purpose embryos in a one cell stage [obtained by crossing hybrid females (CBAxC57BL) to NIH males] were cultured in vitro during 72 hs, individually fixed at morula stage and stained with Feulgen. The DNA content of 263 individual nucleus was evaluated cytophotometrically corresponding to 22 compact morulas of normal development. As haploid PB is present in all pre implanted stage, only embryos with one haploid nuclei were considered as normal. In 95.5% (n = 21) of the embryos the PB was present. DNA measurement of 21 PB was 1n {+-} 0.1. By the height sensibility of PB ploidy, the abnormalities were detected by the criterion of >4.1 n and <1.9 n. The results showed that one embryo was completely haploid (1n). The rest of the embryos (n = 20) 222 blastomeres and 20 PB were analyzed. The DNA measurement showed that 92,7% of the blastomeres (n = 206) are between 2 n and 4 n and 7.3% showed ploidy anomalies, regarding the value n of their PB. The period of the cellular cycle was studied in the normal cell ploidy. This study showed that 16.5% of the blastomeres (n = 34) were in the period G1, 70.39% (n =34) in the period S and 13.2% in the period G2 (n = 27). It is concluded that the PB study showed that it has properties as an excellent indicator of internal ploidia: it is present from the moment of the conception, easily recognizable in the perivitelin space in the embryo of one-two cells, remains in interface during the preimplantation development, it is haploid and digitalized pixel by pixel PB study showed the homogeneity of this type of cell, giving a reliable value of ploidy. The properties of the PB and the results showed that the PB could be an excellent indicator for embryonic ploidy studies on genotoxicity, maintaining its original ploidia during the preimplantation development while the blastomeres are

  12. The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development.

    Science.gov (United States)

    Kang, Sung-Sik; Ofuji, Sosuke; Imai, Kei; Huang, Weiping; Koyama, Keisuke; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2015-06-01

    The aim of the present study was to clarify the efficacy of the well of the well (WOW) culture system for a small number of embryos and the effect of number of adjacent embryos in a WOW dish on blastocyst development. In conventional droplet culture, embryos in the small-number group (5-6 embryos/droplet) showed low blastocyst development compared with a control group (25-26 embryos/droplet). However, small and large numbers of embryos (5-6 and 25 embryos, respectively) in a WOW dish showed no significant differences in cleavage, blastocyst rates, and mean cell number in blastocysts compared with the control group (25-30 embryos/droplet). In addition, the number of adjacent embryos in a WOW dish did not affect the development to blastocysts and cell number in blastocysts. In conclusion, a WOW dish can provide high and stable blastocyst development in small group culture wherever embryos are placed in microwells of the WOW dish.

  13. 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.

  14. Radon-induced cancer: a cell-based model of tumorigenesis due to protracted exposures

    International Nuclear Information System (INIS)

    Elkind, M.M.

    1994-01-01

    In 1982, results with C3H mouse embryo cells showed that the frequency of neoplastic transformation was enhanced when exposures to fission-spectrum neutrons were protracted in time. This finding was unexpected because the opposite was found with low-LET radiations. Similar neutron enhancements were reported with normal life-span Syrian hamster embryo cells, and with human hybrid cells. Because other studies did not confirm the preceding, in 1990 - at a conference convened by the US Armed Forces Radiobiological Research Institute - a biophysical model was proposed to explain the basis for the enhancement observed in some experiments but not in others. The model attributed special sensitivities, related to killing and neoplastic transformation, to cells in and around mitosis. Subsequently, it was shown that late G 2 /M phase cells constituted this window of sensitivity. In the instance of tumorigenesis, the model predicted that protracted exposures to a high-LET radiation would result in enhanced frequencies of transformation providing that susceptible cells were cycling or could be induced to cycle. The model explained data on lung tumour induction in rats breathing radon at different concentrations, and uranium miners working in atmospheres containing different concentrations of radon. The model also explains the anomalous finding that lung cancer deaths are often sublinearly correlated with indoor radon concentration. (author)

  15. The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

    Directory of Open Access Journals (Sweden)

    Hitomi Suzuki

    Full Text Available BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

  16. 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.

  17. 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

  18. The effect of insecticide Deltamethrin on development of chick embryos

    International Nuclear Information System (INIS)

    Al-Naal, R.; Bassal, M. Osman, M.

    1997-04-01

    This study was conducted to evaluate the cyto and the embryo toxicity of Deltamethrin and its commercial formulation DECIS 50 EC in chick embryo during its critical embryonic development period before and in the organogenesis. The embryos were incubated in well closed plastic caps containing the complete egg composition at 38 o. the Deltamethrin and DECIS were found to cause histological and morphological malformations, specially in the brain, also they reduced the majority of the synthetic activities of the DNA, RNA, and proteins in the embryonic and the vascular areas. The flow cytometric analysis showed alterations in frequency of cells in both embryonic and vascular areas in the treated embryo during the cell cycle phases. Our study also showed that the DECIS had greater cyto and embryo toxicity than the Seltamethrin for analysis (author). 149 refs., 36 figs., 16 tabs

  19. Development of Taenia pisiformis in golden hamster (Mesocricetus auratus

    Directory of Open Access Journals (Sweden)

    Maravilla Pablo

    2011-07-01

    Full Text Available Abstract The life cycle of Taenia pisiformis includes canines as definitive hosts and rabbits as intermediate hosts. Golden hamster (Mesocricetus auratus is a rodent that has been successfully used as experimental model of Taenia solium taeniosis. In the present study we describe the course of T. pisiformis infection in experimentally infected golden hamsters. Ten females, treated with methyl-prednisolone acetate were infected with three T. pisiformis cysticerci each one excised from one rabbit. Proglottids released in faeces and adults recovered during necropsy showed that all animals were infected. Eggs obtained from the hamsters' tapeworms, were assessed for viability using trypan blue or propidium iodide stains. Afterwards, some rabbits were inoculated with eggs, necropsy was performed after seven weeks and viable cysticerci were obtained. Our results demonstrate that the experimental model of adult Taenia pisiformis in golden hamster can replace the use of canines in order to study this parasite and to provide eggs and adult tapeworms to be used in different types of experiments.

  20. Zika Virus Exhibits Lineage-Specific Phenotypes in Cell Culture, in Aedes aegypti Mosquitoes, and in an Embryo Model

    Directory of Open Access Journals (Sweden)

    Katherine A. Willard

    2017-12-01

    Full Text Available Zika virus (ZIKV has quietly circulated in Africa and Southeast Asia for the past 65 years. However, the recent ZIKV epidemic in the Americas propelled this mosquito-borne virus to the forefront of flavivirus research. Based on historical evidence, ZIKV infections in Africa were sporadic and caused mild symptoms such as fever, skin rash, and general malaise. In contrast, recent Asian-lineage ZIKV infections in the Pacific Islands and the Americas are linked to birth defects and neurological disorders. The aim of this study is to compare replication, pathogenicity, and transmission efficiency of two historic and two contemporary ZIKV isolates in cell culture, the mosquito host, and an embryo model to determine if genetic variation between the African and Asian lineages results in phenotypic differences. While all tested isolates replicated at similar rates in Vero cells, the African isolates displayed more rapid viral replication in the mosquito C6/36 cell line, yet they exhibited poor infection rates in Aedes aegypti mosquitoes compared to the contemporary Asian-lineage isolates. All isolates could infect chicken embryos; however, infection with African isolates resulted in higher embryo mortality than infection with Asian-lineage isolates. These results suggest that genetic variation between ZIKV isolates can significantly alter experimental outcomes.