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‣ Mutagenicity and Antimutagenicity of Baccharis dracunculifolia Extract in Chromosomal Aberration Assays in Chinese Hamster Ovary Cells

MUNARI, Carla Carolina; RESENDE, Flavia Aparecida; ALVES, Jacqueline Morais; SOUSA, Joao Paulo Barreto de; BASTOS, Jairo Kenupp; TAVARES, Denise Crispim
Fonte: GEORG THIEME VERLAG KG Publicador: GEORG THIEME VERLAG KG
Tipo: Artigo de Revista Científica
Português
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Baccharis dracunculifolia De Candole (Asteraceae), a native plant from the Brazilian ""cerrado"", is widely used in folk medicine as an anti-inflammatory agent and for the treatment of gastrointestinal diseases. B. dracunculifolia has been described as the most important plant source of propolis in southeastern Brazil, which is called green propolis due to its color. The aim of the present study was to evaluate the mutagenic and antimutagenic effects of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE) on Chinese hamster ovary cells. On one hand, the results showed a significant increase in the frequencies of chromosome aberrations at the highest Bd-EAE concentration tested (100 mu g/mL). On the other hand, the lowest Bd-EAE concentration tested (12.5 mu/mL) significantly reduced the chromosome damage induced by the chemotherapeutic agent doxorubicin. The present results indicate that Bd-EAE has the characteristics of a so-called Janus compound, that is, Bd-EAE is mutagenic at higher concentrations, whereas it displays a chemopreventive effect on doxorubicin-induced mutagenicity at lower concentrations. The constituents of B. dracunculifolia responsible for its mutagenic and antimutagenic effects are probably flavonoids and phenylpropanoids...

‣ Continuous and High-Level In Vivo Delivery of Endostatin From Recombinant Cells Encapsulated in TheraCyte (R) Immunoisolation Devices

MALAVASI, N. V.; RODRIGUES, D. B.; CHAMMAS, R.; CHURA-CHAMBI, R. M.; BARBUTO, J. A. M.; BALDUINO, K.; NONOGAKI, S.; MORGANTI, L.
Fonte: COGNIZANT COMMUNICATION CORP Publicador: COGNIZANT COMMUNICATION CORP
Tipo: Artigo de Revista Científica
Português
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Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte (R) system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte (R) devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte (R) were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 mu g/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 mu g/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it...

‣ Lycopene activity against chemically induced DNA damage in Chinese hamster ovary cells

Scolastici, C.; Alves de Lima, R. O.; Barbisan, L. F.; Ferreira, A. L.; Ribeiro, D. A.; Salvadori, Daisy Maria Favero
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 840-845
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Lycopene is a natural pigment synthesized by plants and microorganisms, and it is mainly found in tomatoes. It is an acyclic isomer of P-carotene and one of the most potent antioxidants. Several studies have demonstrated the ability of lycopene to prevent chemically induced DNA damage; however, the mechanisms involved are still not clear. In the present study, we investigated the antigenotoxic/antimutagenic effects of lycopene in Chinese Hamster Ovary Cells (CHO) treated with hydrogen peroxide, methylmethanesulphonate (MMS), or 4-nitroquinoline-1-oxide (4-NQO). Lycopene (97%), at final concentrations of 10, 25, and 50 M, was tested under three different protocols: before, simultaneously, and after the treatment with the mutagens. Comet and cytokinesis-block micronucleus assays were used to evaluate the level of DNA damage. Data showed that lycopene reduced the frequency of micronucleated cells induced by the three mutagens. However, this chemopreventive activity was dependent on the concentrations and treatment schedules used. Similar results were observed in the comet assay, although some enhancements of primary DNA damage were detected when the carotenoid was administered after the mutagens. In conclusion, our findings confirmed the chemopreventive activity of lycopene...

‣ Fluoride does not induce DNA breakage in Chinese hamster ovary cells in vitro.

Ribeiro, Daniel Araki; Scolastici, Clarissa; Marques, Mariângela Esther Alencar; Salvadori, Daisy Maria Favero
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 192-196
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Fluoride has been widely used in dentistry because it is a specific and effective caries prophylactic agent. However, excess fluoride may represent a hazard to human health, especially by causing injury to genetic material. Genotoxicity tests represent an important part of cancer research to assess the risk of potential carcinogens. In the current study, the potential DNA damage associated with exposure to fluoride was assessed by the single cell gel (comet) assay in vitro. Chinese hamster ovary cells were exposed to sodium fluoride (NaF) at final concentration ranging from 7 to 100 micro/ml for 3 h, at 37 dgrees C. The results pointed out that NaF in all concentrations tested did not contribute to DNA damage as depicted by the mean tail moment and tail intensity. These findings are clinically important since they represent an important contribution to a correct evaluation of the potential health risk associated with the exposure to dental agents.

‣ Microphotometric scanning of chromatid gaps and breaks induced by AluI and BamHI in Chinese hamster ovary cells

Martínez-López,Wilner; Bonomi,Rossana; Folle,Gustavo A.; Drets,Máximo E.
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/1996 Português
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Chromatid gaps and breaks induced by the restriction endonucleases AluI and BamHI in the long arm of chromosome 1 of Chinese hamster ovary cells were microphotometrically scanned and mapped to a quantitative G-band map. More than 50% of chromatid breaks appeared as chromatin losses of greater than 5% of the total arm length. The majority of chromatid gaps and breaks as well as chromatin losses induced by both restriction endonucleases were non-randomly located in a region from 0.35 to 0.65 relative length units of the long arm of chromosome 1. We suggest that the access of these endonucleases to chromosomal DNA depends on the local organization of the chromatin.

‣ A cowpox virus gene required for multiplication in Chinese hamster ovary cells.

Spehner, D; Gillard, S; Drillien, R; Kirn, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1988 Português
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Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus.

‣ Superoxide mediates the toxicity of paraquat for Chinese hamster ovary cells.

Bagley, A C; Krall, J; Lynch, R E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1986 Português
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The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action.

‣ Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells.

Ferrara, N; Winer, J; Burton, T; Rowland, A; Siegel, M; Phillips, H S; Terrell, T; Keller, G A; Levinson, A D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1993 Português
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Vascular endothelial growth factor (VEGF) is a mitogen with a specificity for endothelial cells in vitro and an angiogenic inducer in vivo. We tested the hypothesis that VEGF may confer on expressing cells a growth advantage in vivo. Dihydrofolatereductase--Chinese hamster ovary cells were transfected with expression vectors which direct the constitutive synthesis of VEGF. Neither the expression nor the exogenous administration of VEGF stimulated anchorage-dependent or anchorage-independent growth of Chinese hamster ovary cells in vitro. However, VEGF-expressing clones, unlike control cells, demonstrated an ability to proliferate in nude mice. Histologic examination revealed that the proliferative lesions were compact, well vascularized, and nonedematous. Ultrastructural analysis revealed that capillaries within the lesions were of the continuous type. These findings indicate that the expression of VEGF may confer on cells the ability to grow in vivo in the absence of transformation by purely paracrine mechanisms. Since VEGF is a widely distributed protein, this property may have relevance for a variety of physiological and pathological proliferative processes.

‣ Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells.

Heffernan, M; Dennis, J W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/01/1991 Português
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Eukaryotic expression vectors have been used successfully in viral LT-expressing cell lines (ie. COS) to clone cDNAs encoding proteins that can be detected through their bio-activity or reactivity with specific antibodies. Since Chinese hamster ovary cells (CHO) have been used extensively for the isolation and characterization of somatic cell mutants, we felt it would be an advantage to develop an expression cloning system in CHO cells. We have modified the eukaryotic expression vector CDM8 by replacing the polyoma and SV40 origins of replication with the 427bp non-coding region of the Syrian hamster papovavirus. Wild-type CHO cells and the CHO glycosylation-mutant Lec4A were transfected with plasmids bearing the early genes of either polyoma virus or hamster papovavirus in order to establish stable, LT antigen-expressing cell lines designated CHOP or CHOH, respectively. CHOP cell lines expressing polyoma LT antigen supported efficient replication of CDM8, but replicated pMH poorly. Conversely, CHOH cells expressing the hamster papovavirus LT antigen supported replication of pMH, and at a lower efficiency, CDM8. Replication of CDM8 and pMH vectors were equally efficient in selected CHOP and CHOH cell lines, respectively and comparable to that of CDM8 replication in COS-1 cells. A bacterial beta-galactosidase fusion gene inserted into the multiple cloning site of a CDM8 derivative was efficiently expressed when transiently transfected into CHOP and CHOH cells but not CHO cells since only the former supports autonomous plasmid replication. These results show that expression-cloning in CHO cells expressing either polyoma virus or hamster papovavirus LT antigens is possible using either the CDM8 or the pMH vectors...

‣ Role of DNA repair in mutagenesis of Chinese hamster ovary cells by 7-bromomethylbenz[a]anthracene.

Thompson, L H; Brookman, K W; Carrano, A V; Dillehay, L E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1982 Português
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The role of DNA repair in mutagenesis was studied in normal, repair-proficient Chinese hamster ovary cells and in two mutant strains that are deficient in excision repair. By using the mutagen 7-bromomethylbenz[a]anthracene (7-BrMeBA) and the technique of alkaline elution of DNA, the mutants were found to be defective at or before the incision step of excision repair. Dose--responses were determined for cell killing, mutation induction at three loci, and sister chromatid exchanges over a survival range of 1.0--0.1 after 7-BrMeBA treatment. The mutants were 5-fold more sensitive to killing than were the normal cells, but the degree of hypersensitivity to mutation induction varied depending on the mutant strain, the genetic marker, and the dose of mutagen. In each instance, the dose--response curve for mutations was essentially linear in the repair-deficient cells. In the normal cells, however, the curves for induced resistance to thioguanine and azaadenine were complex and were curvilinear with increasing slope at low doses. This behavior may be attributable to saturation of the excision repair system. No difference was seen in the efficiency of inducing ouabain-resistant mutations in the repair-deficient cells compared to the normal cells...

‣ ESR studies of O2 uptake by Chinese hamster ovary cells during the cell cycle.

Lai, C S; Hopwood, L E; Hyde, J S; Lukiewicz, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1982 Português
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Magnetic interactions between dissolved oxygen and nitroxide radical spin probes lead to broadening of the ESR lines. We have used a closed-chamber method based on this property to determine the maximum rate of O2 uptake per cell (Vmax per cell) in cultured mammalian cells. A suitable spin probe and a cell suspension are mixed in an aerated medium, and the rate of disappearance of dissolved O2 is measured. The effects of temperature, pH, and microwave power on the determination of dissolved oxygen in solution were studied. For asynchronous Chinese hamster ovary cells, oxygen uptake is 3.8 X 10(7) oxygen molecules per cell per sec and appears to be enzymatically limited at oxygen concentrations greater than 10 microM. About 5-10 X 10(5) cells were used for each measurement, making it possible to study mitotically synchronized cells. Using this method, we have found that Vmax per unit cell volume changes during the cell cycle of Chinese hamster ovary cells from a minimum in mitosis to maxima in both G1 and late S phases. Advantages and limitations of spin probes for studying the O2 uptake of intact cells are discussed.

‣ Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells.

Kaufman, R J; Wasley, L C; Davies, M V; Wise, R J; Israel, D I; Dorner, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1989 Português
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In plasma, antihemophilic factor (factor VIII) exists as a 200-kilodalton heavy-chain polypeptide in a metal ion association with an 80-kilodalton light-chain polypeptide. This complex is bound by hydrophobic and hydrophilic interactions to a large multimeric glycoprotein, von Willebrand factor (vWF). Accumulation of secreted human factor VIII activity expressed in Chinese hamster ovary cells requires the addition of serum in the growth medium, which provides vWF. Here we report that coexpression of vWF with factor VIII in Chinese hamster ovary cells resulted in increased stable accumulation of factor VIII activity in the absence of serum in the growth medium. In the coexpressing cells, the vWF cDNA transcription unit was transcribed to yield mRNA which was efficiently translated. vWF was properly processed and secreted to yield disulfide-bonded high-molecular-weight multimers similar to those observed in vWF secreted from human endothelial cells. Nuclear run-on assays showed that the factor VIII gene was transcribed at a level similar to that of the vWF gene, but the mRNA did not accumulate to high levels in the cytoplasm. In addition, although the translation efficiency of the factor VIII mRNA was similar to that of vWF, the processing and secretion of the factor VIII primary translation product was dramatically reduced compared with vWF. These results demonstrate that in Chinese hamster ovary cells both factor VIII mRNA accumulation and the processing and secretion of the primary factor VIII translation product are inefficient processes.

‣ Isolation and partial characterization of a cholesterol-requiring mutant of Chinese hamster ovary cells.

Chang, T Y; Telakowski, C; Heuvel, W V; Alberts, A W; Vagelos, P R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1977 Português
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A sterol-requiring mutant has been isolated from mutagenized Chinese hamster ovary cells. This mutant grows normally only when cholesterol is present in the medium. Cell lysis occurs within 3 days in the absence of cholesterol. The frequency of reversion of this mutant to prototrophic growth is low (less than or equal to 10(-6). Whole cell pulse experiments with [14C]acetate or [3H]mevalonate indicate that the rate of synthesis of digitonin-precipitable material is greatly diminished in the mutant cells as compared to that in normal Chinese hamster ovary cells. Enzyme assays in vitro with crude cell extracts show that the biosynthetic conversion of mevalonate to squalene and the conversion of squalene to lanosterol are not impaired in the mutant cells. Gas-liquid chromatographic analyses of radioactive sterol composition after whole cell pulse experiments with [3H]squalene and with [3H]anosterol suggest that the fundamental enzymatic defect of the mutant is at the stage of lanosterol demethylation. When cells were grown in serum-free medium, lanosterol and dihydrolanosterol accumulated intracellularly in the mutant cells before cell lysis occurred; neither of these two intermediary sterols was detected in the wild-type cells grown under the same condition.

‣ Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/07/1987 Português
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Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor- variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100- fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants...

‣ Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

Hunt, S. M. N.; Pak, S. C. O.; Bridges, M. W.; Gray, P. P.; Sleigh, M. J.
Fonte: Kluwer Academic Publishers Publicador: Kluwer Academic Publishers
Tipo: Artigo de Revista Científica
Publicado em /05/1997 Português
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Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

‣ Variability in the Stability and Productivity of Transfected Genes in Chinese Hamster Ovary (CHO) cells

Ng, Say Kong; Yap, Miranda G.S.; Wang, Daniel I.C.
Fonte: MIT - Massachusetts Institute of Technology Publicador: MIT - Massachusetts Institute of Technology
Tipo: Artigo de Revista Científica Formato: 22895 bytes; application/pdf
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In the field of biologics production, productivity and stability of the transfected gene of interest are two very important attributes that dictate if a production process is viable. To further understand and improve these two traits, we would need to further our understanding of the factors affecting them. These would include integration site of the gene, gene copy number, cell phenotypic variation and cell environment. As these factors play different parts in the development process, they lead to variable productivity and stability of the transfected gene between clones, the well-known phenomenon of “clonal variation”. A study of this phenomenon and how the various factors contribute to it will thus shed light on strategies to improve productivity and stability in the production cell line. Of the four factors, the site of gene integration appears to be one of the most important. Hence, it is proposed that work is done on studying how different integration sites affect the productivity and stability of transfected genes in the development process. For the study to be more industrially relevant, it is proposed that the Chinese Hamster Ovary dhfr-deficient cell line, CHO-DG44, is used as the model system.; Singapore-MIT Alliance (SMA)

‣ Differential expression of three alpha-tubulin genes in Chinese hamster ovary cells.

Elliott, E M; Okayama, H; Sarangi, F; Henderson, G; Ling, V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1985 Português
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Chinese hamster ovary cells contain a complex family of ca. 16 unique alpha-tubulin sequences and a similar multiplicity of beta sequences. To examine which members of this multigene family are expressed, we constructed cDNA libraries from two Chinese hamster ovary cell lines according to the method of H. Okayama and P. Berg (Mol. Cell. Biol. 3:280-289, 1983). Each library consisted of 5.5 X 10(5) transformants and contained a high percentage of full-length tubulin clones. Three different alpha-tubulin genes were identified by sequence analysis of the 3' noncoding regions of these tubulin clones. The relative abundance of the transcripts corresponding to the three genes was estimated by gene-specific dot blotting of 96 cDNA alpha-tubulin clones and was found to be 71, 24, and 5%. There is little homology in the 3' noncoding sequences of these genes; however, a strong interspecies homology exists in this region for two of the Chinese hamster ovary genes with the two alpha-tubulin genes previously described in other systems. The third Chinese hamster ovary gene, with an expression frequency of 24%, is unique in that its 3' noncoding region is unlike that of the other mammalian alpha-tubulin genes. In addition, limited sequence data from the coding region of this gene indicates it codes for a unique alpha-tubulin protein.

‣ Increased sensitivity to oxidative injury in chinese hamster ovary cells stably transfected with rat liver S-adenosylmethionine synthetase cDNA.

Sánchez-Góngora, E; Pastorino, J G; Alvarez, L; Pajares, M A; García, C; Viña, J R; Mato, J M; Farber, J L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/11/1996 Português
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Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerse, but not the antioxidant N,N'-diphenylphenylenediamine, prevented the killing of Chinese hamster ovary cells by H2O2 and maintained the contents of NAD and ATP. The results of this study indicate that a moderate activation of the synthesis of S-adenosylmethionine leads to ATP and NAD depletion and to a greater sensitivity to cell killing by oxidative stress.

‣ Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.

Lau, Y F; Lin, C C; Kan, Y W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1984 Português
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We studied the effects of gene amplification on human globin gene expression in Chinese hamster ovary cells. The normal human alpha-globin gene (N alpha 2) and a hybrid gene (M alpha G) containing the 5' promoter-regulator region of the mouse metallothionein gene and the human structural alpha 2-globin gene were linked to a modular SV2-cDNA dihydrofolate reductase (DHFR) gene. The recombinant DNA molecules were introduced into Chinese hamster ovary cells by calcium phosphate precipitation. After initial selection to retain the DHFR and linked sequences, the cells were cultured in increasing concentrations of methotrexate up to 0.2 mM. Southern blot analysis of total cellular DNA showed an approximately 500- to 1,000-fold increase in the number of copies of DHFR and human alpha-globin genes. The transcription of the alpha-globin and DHFR genes increased as their copy number within the cells increased. The transcription of the amplified hybrid M alpha G gene was also inducible with cadmium treatments. Both mature mRNA and "read-through" transcripts were observed. DHFR constituted approximately 10% of pulse-labeled cellular proteins in these cells, but no human alpha-globin was detected. In vitro translation of polyadenylated RNA from these cells showed that alpha-globin mRNA transcribed from the amplified alpha-globin genes was functional and directed alpha-globin chain synthesis. In situ hybridization of 3H-labeled alpha-globin and DHFR DNA probes in chromosome preparations from the two cell lines indicated that both genes were coamplified in the same chromosomal locations in each cell type. These results indicate that gene amplification enhances human globin gene expression in cultured Chinese hamster ovary cells.

‣ Modeling the Effect of Ammonia on the Glycosylation Pattern of Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Adadevoh, Joanna
Fonte: University of Delaware Publicador: University of Delaware
Tipo: Tese de Doutorado
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Babatunde A. Ogunnaike; Monoclonal antibodies (mAbs) are therapeutic recombinant proteins which are typically produced industrially by Chinese Hamster Ovary Cells (CHO). In order to approve mAbs for therapeutic purposes, regulatory agencies require manufacturers to assure the bioactivity, purity, and potency of these proteins to consumers. Regulatory agencies now encourage manufacturers to develop strategies to control quality attributes online. One of the most important factors affecting the quality and bioactivity of mAbs is glycosylation ??? the addition of a carbohydrate chain to a protein. Glycosylation is a post-translational modification which exhibits heterogeneity that arises from variations in site occupancy and structure of attached glycans. As a result of such heterogeneity, glycosylation presents a challenge to manufacturers of mAbs yet an on-line control strategy does not exist for glycosylation. While a number of culture conditions have been found to influence mAb glycoform distribution, in order to develop effective control schemes, it is important to understand the mechanisms underlying how these culture conditions affect glycosylation. One such culture condition is ammonia levels in culture media. Ammonia has been found to adversely affect cell growth and viability. It is also known to induce heterogeneity in glycosylation by affecting the extent of terminal sialylation...