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1.
Jia Dong Carl-Fredrik Mandenius Marc Lübberstedt Thomas Urbaniak Andreas K. N. Nüssler Daniel Knobeloch Jörg C. Gerlach Katrin Zeilinger 《Cytotechnology》2008,57(3):251-261
Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving
cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth
of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according
to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte
growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell
line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth
factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in
metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult.
Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects
on the metabolic functions of primary human hepatocytes. 相似文献
2.
Whether chicken Mx inhibits influenza virus replication is an important question with regard to strategies aimed at enhancing influenza resistance in domestic flocks. The Asn631 polymorphism of the chicken Mx protein found in the Shamo (SHK) chicken line was previously reported to be crucial for the antiviral activity of this highly polymorphic chicken gene. Our aims were to determine whether cells from commercial chicken lines containing Asn631 alleles were resistant to influenza virus infection and to investigate the effects that other polymorphisms might have on Mx function. Unexpectedly, we found that the Asn631 genotype had no impact on multicycle replication of influenza virus (A/WSN/33 [H1N1]) in primary chicken embryo fibroblast lines. Furthermore, expression of the Shamo (SHK) chicken Mx protein in transfected 293T cells did not inhibit viral gene expression (A/PR/8/34 [H1N1], A/Duck/England/62 [H4N6], and A/Duck/Singapore/97 [H5N3]). Lastly, in minireplicon systems (A/PR/8/34 and A/Turkey/England/50-92/91 [H5N1]), which were highly sensitive to inhibition by the murine Mx1 and human MxA proteins, respectively, Shamo chicken Mx also proved ineffective in the context of avian as well as mammalian cell backgrounds. Our findings demonstrate that Asn631 chicken Mx alleles do not inhibit influenza virus replication of the five strains tested here and efforts to increase the frequency of Asn631 alleles in commercial chicken populations are not warranted. Nevertheless, chicken Mx variants with anti-influenza activity might still exist. The flow cytometry and minireplicon assays described herein could be used as efficient functional screens to identify such active chicken Mx alleles. 相似文献
3.
Synthesis of Sindbis virus nonstructural polypeptides in chicken embryo fibroblasts. 总被引:10,自引:10,他引:0
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The identification of eight previously undescribed polypeptides in chicken embryo cells infected with Sindbis virus is reported. Seven of these polypeptides were distinguishable from the virus structural polypeptides and their precursors by their molecular weights and tryptic peptide maps. The eighth was closely related to pE2 (Schlesinger and Schlesinger, 1973), a precursor to one of the virus particle glycoproteins. Pulse-chase experiments and the use of an inhibitor of proteolytic cleavage allowed a division of the seven nonstructural (NS) polypeptides into three stable end products (NS p89, NS p82, and NS p60) and four precursors (p230, p215, p150, and p76). The labeling kinetics after synchronous initiation of translation indicated that synthesis of the NS polypeptides started at a single site and showed that the order of the genes coding for the NS polypeptides was (5' leads to 3') NS p60, NS p89, and NS p82. Short-pulse experiments under conditions of both synchronized and nonsynchronized translation suggested that cleavage of the primary translation product of the NS genes occurred only after its synthesis was completed and that the first cleavage removed the C-terminal polypeptide. From these and other experiments, we propose a detailed scheme for the synthesis and processing of Sindbis virus NS polypeptides. 相似文献
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Juno Jang Sung-Hwan Hong Dongwon Choi Kang-Seuk Choi Seongman Kang Ik-Hwan Kim 《Applied microbiology and biotechnology》2010,85(5):1509-1520
Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential
as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production
system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent
manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked
efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established.
The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had
no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase
in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or
expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the
early phase of viral infection and will be useful in improving the production of viral vaccine strains. 相似文献
6.
Transformation of Brown Leghorn chicken embryo fibroblasts by avian myeloblastosis virus proviral DNA. 总被引:3,自引:1,他引:2
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Brown Leghorn chicken embryo fibroblasts were transfected with a mixture of avian myeloblastosis virus (AMV) and myeloblastosis-associated virus type 1 (MAV1) proviral DNA purified from lambda-Charon 4A recombinant clones. A transformed cell line (T1AM) able to grow without anchorage in semisolid medium was obtained. The presence of both proviral AMV and MAV sequences was detected in T1AM DNA by hybridization with v-myb- and MAV1-specific probes. Altered AMV and MAV1 proviral genomes were found in T1AM genome. Characterization of the RNA species expressed in transformed cells showed that in addition to a 2.5-kilobase (kb) putative subgenomic v-myb-specific RNA, three other myb-containing RNAs (9.4, 8.4, and 7.0 kb) were present in T1AM cells. No AMV genomic RNA was detected. Also, a new 5.0-kb MAV1-specific RNA species was expressed in transformed cells in addition to MAV1 genomic RNA species (7.8 kb). No infectious AMV virions are released by T1AM cells. Chicken embryo fibroblasts infected by T1AM-released virions contained and expressed all MAV1 sequences detected in T1AM transformed cells but did not express any transformation parameter. These results indicated that the presence of AMV proviral sequences in T1AM cells is responsible for their transformed phenotype. 相似文献
7.
Enhanced amino acid transport is observed when quiescent cultures of chicken embryo fibroblasts are stimulated to proliferate by the addition of purified multiplication-stimulating activity (MSA). This increase in amino acid transport is an early event occuring prior to the onset of DNA synthesis in stimulated cells. Results indicate that the changes in transport activity, as measured by α-aminoisobutyric acid (AIB) uptake, are due to stimulation of only the Na+-dependent A transport system. There is little or no change in the activities of transport systems ASC, L, or Ly+ upon exposure to MSA. A kinetic analysis shows this increased activity is due to a change in Vmax while Km remains unaltered. Continuous exposure to the stimulus is required to maintain the increased level of transport activity and the presence of inhibitors of RNA and protein synthesis significantly inhibits the response. Results also indicate that a similar specific increase in the A transport system is initiated when RSV tsNY68 infected cells are shifted to the permissive temperature. It appears that the A system of mediation is emerging as a strategic regulatory site for cell function. 相似文献
8.
Independent expression of avian sarcoma virus in doubly infected chicken embryo fibroblasts. 总被引:2,自引:0,他引:2
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Infection of a chicken cell with avian sarcoma virus requires division of the infected cell before synthesis of infectious progeny is initiated. This requirement for a cell division for the complete expression of avian sarcoma virus has been examined further with chicken embryo fibroblasts infected with two distinct viruses. Chicken cells infected with and producing a mutant of Rous sarcoma virus temperature sensitive for transformation (tsLA24PR-A) were arrested in G0 by depletion of serum factors from growth medium. These stationary cells continued to produce infectious progeny in the absence of further cell division. Superinfection of the stationary cells with the wild-type Prague strain of Rous sarcoma virus (PR-RSV-C) produced a stable double infection in these cells. Progeny of the superinfecting PR-RSV-C, however, were not detected until these cells underwent division after stimulation with fresh serum-containing medium. The addition of colchicine to these serum-stimulated cells, although not affecting production of the tsLA24PR-A, inhibited the appearance of progeny of the superinfecting PR-RSV-C. These experiments indicate that each avian sarcoma virus infection of a chicken embryo fibroblast requires division of the infected cell for production of that virus regardless of whether or not the cell is already producing a similar virus. The results suggest, therefore, that the requirement for a cell division represents a requirement for an event that controls virus expression in a "cis-acting" fashion specific for the provirus. 相似文献
9.
Replication of modified vaccinia virus Ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene E3L
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Hornemann S Harlin O Staib C Kisling S Erfle V Kaspers B Häcker G Sutter G 《Journal of virology》2003,77(15):8394-8407
Highly attenuated modified vaccinia virus Ankara (MVA) serves as a candidate vaccine to immunize against infectious diseases and cancer. MVA was randomly obtained by serial growth in cultures of chicken embryo fibroblasts (CEF), resulting in the loss of substantial genomic information including many genes regulating virus-host interactions. The vaccinia virus interferon (IFN) resistance gene E3L is among the few conserved open reading frames encoding viral immune defense proteins. To investigate the relevance of E3L in the MVA life cycle, we generated the deletion mutant MVA-DeltaE3L. Surprisingly, we found that MVA-DeltaE3L had lost the ability to grow in CEF, which is the first finding of a vaccinia virus host range phenotype in this otherwise highly permissive cell culture. Reinsertion of E3L led to the generation of revertant virus MVA-E3rev and rescued productive replication in CEF. Nonproductive infection of CEF with MVA-DeltaE3L allowed viral DNA replication to occur but resulted in an abrupt inhibition of viral protein synthesis at late times. Under these nonpermissive conditions, CEF underwent apoptosis starting as early as 6 h after infection, as shown by DNA fragmentation, Hoechst staining, and caspase activation. Moreover, we detected high levels of active chicken alpha/beta IFN (IFN-alpha/beta) in supernatants of MVA-DeltaE3L-infected CEF, while moderate IFN quantities were found after MVA or MVA-E3rev infection and no IFN activity was present upon infection with wild-type vaccinia viruses. Interestingly, pretreatment of CEF with similar amounts of recombinant chicken IFN-alpha inhibited growth of vaccinia viruses, including MVA. We conclude that efficient propagation of MVA in CEF, the tissue culture system used for production of MVA-based vaccines, essentially requires conserved E3L gene function as an inhibitor of apoptosis and/or IFN induction. 相似文献
10.
Fanny Knöspel Rudolf K. Schindler Marc Lübberstedt Stephanie Petzolt Jörg C. Gerlach Katrin Zeilinger 《Cytotechnology》2010,62(6):557-571
The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture
media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells,
the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of
a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis
according to Plackett–Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence
on the proliferation activity of the cells, while zinc and l-cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following
factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays
are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media
for human embryonic stem cells (hESC). 相似文献
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A temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus-infected chicken embryo fibroblasts arrested by serum starvation at the non-permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2-deoxyglucose uptake, 3H-thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication-stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum-free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product. 相似文献
13.
Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus 总被引:4,自引:0,他引:4
Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur. 相似文献
14.
《微生物学免疫学进展》2020,(1)
目的评估自主研发培养基QS作为首选培养基用于冻干人用狂犬病疫苗(鸡胚成纤维细胞)生产过程的可行性。方法分别制备基于自主研发培养基QS和其他3种商业化培养基(X1、X2、X3)的鸡胚成纤维细胞悬液,观察和比较细胞形态和生长特性;以Flury HEP株接种鸡胚成纤维细胞(MOI=0.003),分别通过直接免疫荧光法、蛋白质印迹法(Western blotting)和酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)比较不同培养基条件下收获液中病毒滴度和G蛋白含量。结果在细胞浓度1×10~6个/mL条件下,4种培养基培养的鸡胚成纤维细胞在形态上均无显著区别;但是自主研发培养基QS和X2的病毒收获液的G蛋白含量在第4天时分别为0.66 IU/mL和0.63 IU/mL,高于X1的0.5 IU/mL和X3的0.3 IU/mL。在第6天时分别为0.92 IU/mL和0.88 IU/mL,高于X1的0.64 IU/mL和X3的0.52 IU/mL,说明基于自主研发培养基QS和X2的病毒收获液在G蛋白含量方面具有明显优势。结论自主研发培养基QS可以用于冻干人用狂犬病疫苗(鸡胚成纤维细胞)的生产。 相似文献
15.
Avidin, a high-affinity biotin-binding protein of chicken oviduct, was recently found to be synthesized and secreted by damaged or virus-transformed chicken embryo fibroblasts and by chicken macrophages. We have now localized avidin in fibroblasts that were transformed by Rous sarcoma virus. The cells released to the culture medium up to 12 micrograms avidin per 10(6) cells, as judged by the [14C] biotin-binding method. In immunofluorescence microscopy, avidin was localized to the cytoplasm of transformed and of untransformed damaged cells. Treatment with the ionophore monensin was used to determine whether avidin is processed through the Golgi region, which was localized using rhodamine-labeled wheat germ agglutinin. Under these conditions avidin was largely confined to the Golgi region. At the electron microscopic level avidin could be localized to the endoplasmic reticulum of transformed cells, using anti-avidin antibodies and the avidin-biotin-peroxidase complex (ABC) technique. Biotinyl peroxidase did not stain the endogenous avidin in cell layers processed for light or electron microscopy indicating that its biotin-binding sites were either saturated or denaturated. The possibility that endogenous avidin in tissues or cell cultures may bind biotinylated reagents should be controlled for in techniques involving the avidin-biotin interaction. 相似文献
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Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus. 总被引:1,自引:0,他引:1
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In chicken embryo fibroblasts, phosphorylation of the 40S ribosomal protein S6 increases during G1 but returns to basal level by mitosis. In contrast, in Rous sarcoma virus (RSV)-transformed fibroblasts, S6 remains highly phosphorylated throughout mitosis. This study investigated the mechanism by which RSV alters the pattern of S6 phosphorylation. Pulse-chase experiments demonstrate that phosphate turnover in S6 is rapid in normal cells and in cells infected with an RSV transformation-defective virus. In contrast, phosphate turnover in S6 is severely reduced in cells infected with temperature-sensitive RSV at a temperature permissive for transformation, indicating a diminished S6 phosphatase activity. Fractionation of cell lysates by DEAE chromatography showed an almost threefold lower S6 phosphatase activity in RSV-transformed versus normal cells. The S6 phosphatase was sensitive to inhibitor 2 and specifically recognized by an antibody to type 1 phosphatase (PP1). The S6 phosphatase activity recovered by immunoprecipitation of PP1 was threefold lower in transformed cells, but the steady-state level of expression and the rate of synthesis of PP1 were not altered by oncogenic transformation. Together, the results show that transformation by RSV reduced the S6-PP1 activity. 相似文献
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Rate of virus-specific RNA synthesis in synchronized chicken embryo fibroblasts infected with avian leukosis virus.
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The rate of avian leukosis virus (ALV)-specific RNA synthesis has been examined in bot- uninfected and ALV-infected synchronized chicken embryo fibroblasts. RNA from cells labeled for 2h with [3H]uridine was hybridized with avian myeloblastosis virus poly(dC)-DNA, and the hybridized RNA was analyzed with poly(I)-spephadex chromatography. Approximately 0.5% of the RNA synthesized in ALV-infected cells was detected as virus specific, and no more than a twofold variation in the rate of synthesis was detected at different times in the cell cycle. In synchronized uninfected chicken embryo fibroblasts, approximately 0.03% of the RNA synthesized was detected as virus specific, and no significant variation in the rate of synthesis was observed during the cell cycle. Treatment of ALV-infected chicken embryo fibroblasts with cytosine arabinoside or colchicine was used to block cells at different stages in the cell cycle. The rates of virus-specific RNA synthesis in cells so treated did not differ significantly from the rates in either stationary or unsynchronized virus-infected chicken embryo fibroblasts. These findings support the conclusion that after the initial division of an ALV-infected chicken embryo fibroblast and the initiation of virus RNA synthesis, the rate of virus-specific RNA synthesis is independent of the cell cycle. 相似文献