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  • 1
    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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  • 2
    Electronic Resource
    Electronic Resource
    Development and characterization of a rainbow trout liver cell line expressing cytochrome P450-dependent monooxygenase activity (1993)
    Springer
    Cell biology and toxicology 9 (1993), S. 279-294 
    Details
    ISSN: 1573-6822
    Keywords: cytochrome-P450 ; 7-ethoxyresorufinO-deethylase ; trout liver cell line
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A cell line RTL-W1, has been developed from the normal liver of an adult rainbow trout by proteolytic dissociation of liver fragments. RTL-W1 can be grown routinely in the basal medium, L-15, supplemented with 5% fetal bovine serum. In this medium, the cells have been passaged approximately 100 times over an 8-year period. The cells do not form colonies or grow in soft agar. The cultures are heteroploid. The cell shape was predominantly polygonal or epithelial-like, but as cultures became confluent, bipolar or fibroblast-like cells appeared. Among the prominent ultrastructural features of RTL-W1 were distended endoplasmic reticulum and desmosomes. Benzo[a]pyrene was cytotoxic to RTL-W1. Activity for the enzyme, 7-ethoxyresorufin O-deethylase (EROD), which is a measure of the cytochrome P4501A1 protein, increased dramatically in RTL-W1 upon their exposure to increasing concentrations of either β-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). With these properties, RTL-W1 should be useful for studying the expression of the cytochrome P450 enzymes and as a tool for assessing the toxic potency of environmental contaminants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00755606
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  • 3
    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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  • 4
    Electronic Resource
    Electronic Resource
    Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts (1988)
    Springer
    Journal of comparative physiology 158 (1988), S. 457-467 
    Details
    ISSN: 1432-136X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00691143
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  • 5
    Electronic Resource
    Electronic Resource
    Induced thermotolerance to apoptosis in a human T lymphocyte cell line (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 151 (1992), S. 561-570 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A brief exposure to elevated temperatures elicits, in all organisms, a transient state of increased heat resistance known as thermotolerance. The mechanism for this thermotolerant state is unknown primarily because it is not clear how mild hyperthermia leads to cell death. The realization that cell death can occur through an active process of self destruction, known as apoptosis, led us to consider whether thermotolerance provides protection against this mode of cell death. Apoptosis is a common and essential form of cell death that occurs under both physiological and pathological conditions. This mode of cell death requires the active participation of the dying cell and in this way differs mechanistically from the alternative mode of cell death, necrosis. Here we show that mild hyperthermia induces apoptosis in a human leukemic T cell line. This is evidenced by chromatin condensation, nuclear fragmentation and the cleavage of DNA into oligonucleosome size units. DNA fragmentation is a biochemical hallmark of apoptosis and requires the activation of an endogenous endonuclease. The extent of DNA fragmentation was proportional to the severity of heat stress for cells heated at 43°C from 30 to 90 minutes. A brief conditioning heat treatment induced a resistance to apoptosis. This was evident as a resistance to DNA fragmentation and a reduction in the number of apoptotic cells after a heat challenge. Resistance to DNA fragmentation developed during a recovery period at 37°C and was correlated with enhanced heat shock protein (hsp) synthesis. This heat-induced resistance to apoptosis suggests that thermotolerant cells have gained the capacity to prevent the onset of this pathway of self-destruction. An examination of this process in heated cells should provide new insights into the molecular basis of cellular thermotolerance. © 1992 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041510316
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  • 6
    Electronic Resource
    Electronic Resource
    Changes in heat shock protein synthesis and heat sensitivity during mouse thymocyte development (1993)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 14 (1993), S. 148-158 
    Details
    ISSN: 0192-253X
    Keywords: Apoptosis ; DNA fragmentation ; T-cell development ; heat shock proteins ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Heat shock protein synthesis was examined in mouse thymocytes at three stages of development: early embryonic thymocytes, which are CD4-CD8-, adult thymocytes, which are primarily CD4+CD8+, and mature spleen T cells, which are CD4+CD8- or CD4-CD8+. After either a 41°C or 42°C heat shock, the synthesis of the maior heat-inducible protein (hsp68) was elevated during the first hour of recovery but then decreased abruptly in thymocytes from adult mice. In contrast, the synthesis of hsp68 continued for up to 4 h after heating embryonic mouse thymocytes or mature spleen T cells. The more rapid termination ofthe heat shock response in the adult thymocytes was not the result of eitherless heat damage or more rapid repair since the recovery of general protein synthesis was more severely delayed in these cells. As well, the double positive CD4+CD8+ cells were more sensitive to hyperthermia than either the double negative CD4-CD8- or single positive CD4+CD8- or CD4-CD8+ cells. Exposure of fetal thymus organ cultures to elevated temperature revealed that the double negative thymocytes were able to survive and differentiate normally following a heat shock treatment that was lethal for the double positive thymocytes. Exposure of thymocytes from adult mice to elevated temperatures induced apoptotic cell death. This was evident by the cleavage of DNA into oligonucleosome-sized fragments. Quantitation of the extent of DNA fragmentation and the number of apoptotic cells by flow cytometry demonstrated that the extent of apoptotic cell death was related to the severity of the heat stress. Double positive (CD4+CD8+) thymocytes are selected on the basis of their T-cell antigen receptor (TCR). Most of these cells are negatively selected and die within the thymus by an active process of cell deletion known as apoptosis. Restricting hsp synthesis in response to stress might be essential during developmental processes in which cell maturation is likely to result in death rather than functional differentiation. © 1993Wiley-Liss, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020140209
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