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  • 1
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Company
    Nature biotechnology 7 (1989), S. 50-54 
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] There are many commercially and scientifically relevant products that must be produced in mammalian cell systems. Growth and maintenance systems (bioreactors) for mammalian cells are not well characterized, however, since these cells are more fastidious and sensitive than are microorganisms. ...
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  • 2
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 32 (1988), S. 983-992 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A high surface area hollow fiber reactor was developed for mammalian cell culture. The reactor employs an interfiber gel matrix of agar or collagen for cell support. A model was developed to predict cell density as a function of fiber spacing. Optimum spacings are calculated for two sizes of Celgard hollow fibers. Ehrlich Ascites Tumor (EAT) cells were grown to an estimated density of 1.1 × 108 viable cells/mL in the extracapillary space - corresponding to an overall reactor density of 7 × 107 cells/mL. On the basis of available kinetic and diffusivity data, the model predicts that lactate accumulation may limit cell growth in the early stage of medium utilization, while oxygen delivery becomes limiting at later stages.
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  • 5
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of intracellular pH (pHin) in the regulation of cell growth in both normal and transformed cells is a topic of considerable controversy. In an effort to study this relationship NIH 3T3 cells were stably transfected with the gene for the yeast H+-ATPase, constitutively elevating their pHin. The resulting cell line, RN1a, has a transformed phenotype: The cells are serum independent for growth, clone in soft agar, and form tumors in nude mice. In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum-independent growth, using defined media to investigate the effects of specific growth factors on the transfected and parental NIH 3T3 cells. While both cell lines show similar growth increases in response to platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF), they respond differently to insulin, insulin-like growth factor-I (IGF-I) and PDGF-AA. RN1a cells exhibit increased growth at nanomolar concentrations of insulin but the parental cells had only a relatively minor response to insulin at 10 μM. Both cell lines showed some response to IGF-I in the nanomolar range but the response of RN1a cells was much larger. Differences in insulin and IGF-I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF-AA. RN1a cells are relatively insensitive to stimulation by PDGF-AA and express fewer PDGF α receptors as shown by Northern blots and receptor-binding studies. We propose a unifying hypothesis in which the H+-ATPase activates a downstream element in the PDGF-AA signal transduction pathway that complements insulin and IGF-I signals, while leading to downregulation of the PDGF α receptor. © 1994 wiley-Liss, Inc.
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  • 6
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: NIH-3T3 cells transfected with yeast H+-ATPases (RN1A cells) are tumorigenic (Perona and Serrano, 1988, Nature, 334: 438). We have previously shown that RN1a cells maintain a chronically high intracellular pH (pHin) under physiological conditions. We have alsoshown that RN1a cells are serum-independent for growth, maintain a higher intracellular Ca2+(in), and glycolyze more rapidly than their non-transformed counterparts (Gillies et al., Proc. Natl. Acad. Sci., 1990, 87: 7414; Gillies et al., Cell. Physiol. Biochem., 1992, 2: 159). The present study was aimed to understand the interrelationships between glycolysis, pHin, and [Ca2+]in in RN1a cells and their non-transformed counterparts, NIH-3T3 cells. Our data show that the higher rate of glycolysis observed in RN1a cell is due to the presence of low affinity glucose transporters. Consequently, the higher rate of glycolysis is exacerbated at high glucose concentration in RN1a cells. Moreover, the maximal velocity (Vmax) for glucose utilization is up to sixfold higher in RN1a cells than in the NIH-3T3 cells, suggesting that the number of glucose transporters is higher in RN1a than NIH-3T3 cells. Glucose addition to NIH-3T3 cells results in modest decreases in both pHin and [Ca2+]in. In contrast, RN1a cells respond to glucose with a large decrease in pHin, followed by a large decrease in [Ca2+]in. The decrease in [Ca2+]in observed upon glucose addition is likely due to activation of Ca2+-ATPase by glycolysis, since the Ca2+ decrease is abolished by the Ca2+ ATPase inhibitors thapsigargin and cyclopiazonic acid. Glucose addition to ATP-depleted cells results in a decrease in [Ca2+]in, suggesting that ATP furnished by glycolysis is utilized by this pump. © 1994 Wiley-Liss, Inc.
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  • 7
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In a companion paper (Gillies et al.: J. Cell. Physiol. 139:124-129, 1989) we show that phorbol esters (PEs) are unable to stimulate Na+/H+ exchange in BALB/C-3T3 cells under a wide variety of conditions. The Na+/H+ exchangers of a number other cell types are also not responsive to PEs yet have been rendered responsive by treatment with agents such as dimethylsulfoxide (DMSO). We undertook the present study to evaluate whether or not the treatment of BALB/c-3T3 cells with DMSO will induce modifications in the sensitivity of these cells to activation by PEs. The present study indicates that a 3-5 day exposure of BALB/c-3T3 cells to 1.25% DMSO leads to changes in the sensitivity of these cells to the activation of Na+/H+ exchanger by PEs. These changes in sensitivity were apparent at day 3 and maximal at day 5. Non-tumor-promoting analogues of PEs do not activate Na+/H+ exchange, suggesting that the effect is mediated through kinase C. Sphingosine prevents PE-, but not serum-induced alkalinization. However, the half-time of the intracellular pH (pHin) response to serum was increased by sphingosine, suggesting that kinase C participates in, but is not required for the serum induced activation. Since DMSO does not induce any apparent morphological change, the change in sensitivity of Na+/H+ exchange to PEs is not likely to be related to differentiation, but may be associated with structural changes in the Na+/H+ exchanger and/or changes in isoforms of kinase C which recognize the exchanger as a substrate.
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  • 8
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Addition of serum to quiescent mammalian cells in culture initiates a series of events which culminates in DNA replication and cell division. One of the earliest events in this sequence of events is activation of Na+/H+ exchange, which can result in an increase in intracellular pH (pHin). The regulation of this change in activity is not known. Since treatment of 3T3 cells with activators of protein kinase C (kinase C) can result in an increased pHin, it has been hypothesized that serum stimulation of kinase C is responsible for activation of Na+/H+ exchange. Recently, sphingolipids have been discovered to inhibit kinase C both in vitro and in vivo. Therefore, we undertook the present study to ask whether or not inhibition of kinase C using sphingolipids prevents mitogen-induced alkalinization in 3T3 cells. Our results indicate that activators of kinase C stimulate Na+/H+ exchange in normal human fibroblasts (BoGi), but not in mouse embryo (3T3) cells. Addition of serum to BoGi cells, on top of saturating doses of phorbol 12-myristate 13-acetate (PMA), results in a further cytoplasmic alkalinization. Furthermore, sphingosine prevents the PMA-induced increase in pHin in BoGi cells, and phosphorylation of an 80 kDa protein in 3T3 cells, but not the serum-induced alkalinization in either BoGi or 3T3 cells. These data indicate that activation of kinase C does not participate in the physiological activation of Na+/H+ exchange in human fibroblasts or mouse embryo cells by serum.
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  • 9
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    ISSN: 1523-9829
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Technology , Medicine
    Notes: Magnetic resonance spectroscopy (MRS) has been used for more than two decades to interrogate metabolite distributions in living cells and tissues. Techniques have been developed that allow multiple spectra to be obtained simultaneously with individual volume elements as small as 1 uL of tissue (i.e., 1 ?? 1 ?? 1 mm3). The most common modern applications of in vivo MRS use endogenous signals from 1H, 31P, or 23Na. Important contributions have also been made using exogenous compounds containing 19F, 13C, or 17O. MRS has been used to investigate cardiac and skeletal muscle energetics, neurobiology, and cancer. This review focuses on the latter applications, with specific reference to the measurement of tissue choline, which has proven to be a tumor biomarker that is significantly affected by anticancer therapies.
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  • 10
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nuclear magnetic resonance (NMR) spectroscopic analysis of whole cells is an important emerging technique for noninvasive and nondestructive monitoring of cell physiology. However, this technique requires extremely high cell densities. Attempts to maintain densities above the carrying capacity of a maintenance system result in the demise of the entire culture. To define conditions for maintaining mammalian cells at high densities for NMR studies, we have designed a bioreactor to operate under defined, oxygen-limited conditions within an NMR spectrometer. The bioreactor utilizes hollow fibers to deliver nutrients and remove wastes from an agitated cell suspension. The mass transfer properties of the fibers with respect to oxygen were determined. Ehrlich Ascites Tumor (EAT) cells were supplied with glutamine as the respiratory carbon source. The maximum viable cell density supported by a given oxygen concentration in the fluid flowing through the fiber lumen was predicted and then confirmed experimentally on the bench and in the spectrometer.
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