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  • 1
    Electronic Resource
    Electronic Resource
    The Transcription Factor E2F1 Modulates Apoptosis of Neurons (2000)
    Oxford UK : Blackwell Science Ltd.
    Journal of neurochemistry 75 (2000), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract : The transcription factor E2F1 is known to mediate apoptosis in isolated quiescent and postmitotic cardiac myocytes, and its absence decreases the size of brain infarction following cerebral ischemia. To demonstrate directly that E2F1 modulates neuronal apoptosis, we used cultured cortical neurons to show a temporal association of the transcription and expression of E2F1 in neurons with increased neuronal apoptosis. Cortical neurons lacking E2F1 expression (derived from E2F1 -/- mice) were resistant to staurosporine-induced apoptosis as evidenced by the significantly lower caspase 3-like activity and a lesser number of cells with apoptotic morphology in comparison with cortical cultures derived from wild-type mice. Furthermore, overexpressing E2F1 alone using replication-deficient recombinant adenovirus was sufficient to cause neuronal cell death by apoptosis, as evidenced by the appearance of hallmarks of apoptosis, such as the threefold increase in caspase 3-like activity and increased laddered DNA fragmentation, in situ endlabeled DNA fragmentation, and numbers of neuronal cells with punctate nuclei. Taken together, we conclude that E2F1 plays a key role in modulating neuronal apoptosis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.2000.0750091.x
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  • 2
    Electronic Resource
    Electronic Resource
    Improved Adenovirus Vector Provides Herpes Simplex Virus Ribonucleotide Reductase R1 and R2 Subunits Very Efficiently (1995)
    [s.l.] : Nature Publishing Company
    Nature biotechnology 13 (1995), S. 602-608 
    Details
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] We have constructed a new adenovirus (Ad) expression vector, pAdBM5, that allows for the production of unprecedented levels of recombinant protein in the human 293 cell line using the Ad expression system. The main feature of this vector is a combination of enhancer sequences that increases the ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/nbt0695-602
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  • 3
    Electronic Resource
    Electronic Resource
    Oligodendrocyte injury in multiple sclerosis: a role for p53 (2003)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 85 (2003), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Multiple sclerosis (MS) is a neurological disorder characterized by myelin destruction and a variable degree of oligodendrocyte death. We have previously shown that overexpression of the transcription factor p53 can induce oligodendrocyte apoptosis. We investigated the mechanism of p53-induced apoptosis using primary cultures of central nervous system-derived adult human oligodendrocytes. Adenovirus-mediated p53 overexpression resulted in up-regulation of the death receptors Fas, DR4 and DR5 with subsequent caspase-mediated apoptosis of the oligodendrocytes. The oligodendrocytes were protected from p53-induced cell death by blocking signaling through Fas and/or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. Although lower levels of p53 did not induce apoptosis, the increase in death receptor expression was sufficient to render the oligodendrocytes susceptible to apoptosis in the presence of exogenous Fas ligand and TRAIL. These ligands are present in the inflammatory milieu of active MS lesions. In situ analysis of active MS lesions revealed increased p53 expression in oligodendrocytes in lesions that featured oligodendrocyte apoptosis and cell loss. Our data provide evidence for a novel role for p53 in the pathogenesis of MS.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.2003.01674.x
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  • 4
    Electronic Resource
    Electronic Resource
    Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses (1999)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 177 (1999), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-γ.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1999.tb13727.x
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  • 5
    Electronic Resource
    Electronic Resource
    New adenovirus vectors for protein production and gene transfer (1998)
    Springer
    Cytotechnology 28 (1998), S. 53-64 
    Details
    ISSN: 1573-0778
    Keywords: adenoviral recombinant ; functional genomics ; gene therapy ; green fluorescent protein ; inducible gene expression ; protein production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10–15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008013211222
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  • 6
    Electronic Resource
    Electronic Resource
    Induction of apoptosis in oxygen-deprived cultures of hybridoma cells (1994)
    Springer
    Cytotechnology 15 (1994), S. 117-128 
    Details
    ISSN: 1573-0778
    Keywords: Animal cell culture ; anoxia ; apoptosis ; cell death ; hybridoma ; hypoxia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions. Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00762386
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  • 7
    Electronic Resource
    Electronic Resource
    Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells (1994)
    Springer
    Cytotechnology 15 (1994), S. 145-155 
    Details
    ISSN: 1573-0778
    Keywords: Adenovirus ; human 293S cells ; recombinant protein ; scale-up ; metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Human 293S cells, a cell line adapted to suspension culture, were grown to 5×106 cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×105 cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×106 cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00762389
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  • 8
    Electronic Resource
    Electronic Resource
    Use of a micromanipulator for high-efficiency cloning of cells co-expressing fluorescent proteins (2000)
    Springer
    Methods in cell science 22 (2000), S. 137-145 
    Details
    ISSN: 1573-0603
    Keywords: Cell cloning ; Dicistronic vectors ; Fluorescence microscopy ; Green Fluorescent Protein ; Quixell™
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The inclusion of the gene encoding the Green Fluorescent Protein (GFP) or its derivatives into dicistronic transfer vectors is a useful method to visually identify cells that have incorporated a specific gene of interest. By combining this approach with the use of a micromanipulator, we have developed a protocol for the one-step isolation of cells expressing a specific transgene from a pool of transfected cells. Target fluorescent cells could be identified and isolated even when they occured at frequencies as low as 1/100,000. The use of Leibowitz L-15 serum-free medium and serum-coated non-charged petri dishes, along with minimal light exposure yielded maximal cell viability and high cloning efficiency (≈ 40%, on average) for a large number of cell lines, both adherent and suspension. Several variations of the basic method are presented, as well as guidelines for the choice of hardware components to implement our cloning workstation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009871029495
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  • 9
    Electronic Resource
    Electronic Resource
    Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions (1998)
    Springer
    Cytotechnology 28 (1998), S. 189-203 
    Details
    ISSN: 1573-0778
    Keywords: apoptosis ; cell cycle ; E1B-19K ; hydroxyurea ; hyperosmosis ; hypertonic ; monoclonal antibodies ; OptiMAbTM ; thymidine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb™. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAb™ accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb™ resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAb™. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAb™ with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008054403470
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  • 10
    Electronic Resource
    Electronic Resource
    High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 36 (1990), S. 1133-1140 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to develop an efficient process for large-scale production of recombinant protein, various factors were studied which affect the productivity of Sf-9 (Spodoptera frugiperda) insect cells when using the baculovirus expression system. It was shown that upon infection with the Bac-BRV6L recombinant baculovirus, the level per cell of VP6 (a bovine rotavirus nucleocapsid protein) would drop 10-fold when host cell density at the time of infection increased from 2 × 106 to 3 × 106 cells/mL. The decrease was found to be totally reversible by culture medium renewal after infection, even when cells were infected at the stationary phase. Recombinant protein production was 4-6 times higher using TNMFH medium supplemented with 10% fetal bovine serum (FBS) than in IPL/41 serum-free medium. Fine-tuning of infection parameters in a 4-L surface-aerated bioreactor resulted in the production of typically 350 mg/L of VP6 protein, representing more than 25% of total cell proteins.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260361108
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