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  • 1
    Electronic Resource
    Electronic Resource
    CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 518-528 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; serum-free medium ; adaptation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. © 1996 John Wiley & Sons, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Ability of different hepatoma cells to metabolize 4-hydroxynonenal (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 11 (1993), S. 79-86 
    Details
    ISSN: 0263-6484
    Keywords: Aldehyde dehydrogenase ; alcohol dehydrogenase ; aldehyde reductase ; glutathione-S-transferase ; hepatoma ; 4-hydroxynonenal ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: 4-Hydroxynonenal (4-HNE), produced during the oxidative lipid breakdown of biological membranes, modulates various biochemical processes in normal liver and in hepatoma cells. It is very probable that the effects of 4-HNE are related to the quantity formed in the cells and the cells' ability to metabolize it. Aldehyde catabolism takes place within the cells through oxidative and reductive enzymes, and through conjugation with intracellular glutathione. In this paper, the various enzymatic pathways involved in the metabolism of 4-HNE were studied in normal hepatocytes and in hepatoma cells. The hepatocyte pathway undergoes a complex variety of change during neoplastic transformation.In hepatoma cells, generally, 4-HNE metabolism was due mainly to aldehyde dehydrogenases, whereas in normal hepatocytes 4-HNE metabolism was mainly due to alcohol dehydrogenase and glutathione-S-transferase. The increase in oxidative enzymes compared to normal tissue was not the same in all types of hepatoma: in HTC hepatoma cells, the enzyme levels were considerably higher; in AH-130 hepatoma cells of Yoshida, they were lower in subcellular particles and similar in the cytosol. Indeed, consumption of externally-added 4-HNE in hepatoma cells was proportional to their content of 4-HNE metabolizing enzymes.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cbf.290110202
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  • 3
    Electronic Resource
    Electronic Resource
    Comparison of two experimental methods for the determination of Michaelis-Menten kinetics of an immobilized enzyme (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 40 (1992), S. 16-24 
    Details
    ISSN: 0006-3592
    Keywords: Michaelis-Menten kinetics ; biocatalyst particles ; oxygen microsensor ; intrinsic kinetics ; modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: For the application of immobilized enzymes, the influence of immobilization on the activity of the enzyme should be Known. This influence can be obtained by determining the intrinsic kinetic parameters of the immobilized enzyme, and by comparing them with the kinetic parameters of the suspended enzyme. This article deals with the determination of the intrinsic kinetic parameters of an agarose-gel bead immobilized oxygen-consuming enzyme: L-lactate 2-monooxygenase. The reaction rate of the enzyme can be described by Michaelis-Menten kinetics. Batch conversion experiments using a biological oxygen monitor, as well as steady-state profile measurements within the biocatalyst particles using an oxygen microsensor, were performed. Two different mathematical methods were used for the batch conversion experiments, both assuming a pseudosteady-state situation with respect to the shape of the profile inside the bead. One of the methods used an approximate relation for the effectiveness factor for Michaelis-Menten kinetics which interpolates between the analytical solutions for zero- and first-order kinetics. The other mathematical method was based on a numerical solution and combined a mass balance over the reactor with a mass balance over the bead. The main difference in the application of the two methods is the computer calculation time; the completely numerical calculation procedure was about 20 times slower than the other calculation procedure.The intrinsic kinetic parameters resulting from both experimental methods were compared to check the reliability of the methods. There was no significant difference in the intrinsic kinetic parameters obtained from the two experimental methods. By comparison of the kinetic parameters for the suspended enzyme with the intrinsic kinetic parameters for the immobilized enzyme, it appeared that immobilization caused a decrease in the value of Vm by a factor of 2, but there was no significant difference in the values obtained for Km.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260400104
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  • 4
    Electronic Resource
    Electronic Resource
    Effect of cDNA copy number on secretion rate of activated protein C (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 22-27 
    Details
    ISSN: 0006-3592
    Keywords: cDNA copy number ; gene dosage ; recombinant protein production ; posttranslational modification ; BHK ; secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The secretion rate of activated protein C (APC) by BHK cells was increased 35-fold by increasing the cDNA copy number per cell from 50 to 240. In this range, the relation between APC secretion and cDNA copy number was not linear and the rate of APC secretion per cDNA copy increased sevenfold. This apparent cooperative effect of multiple cDNA copies could be related to their integration in tandem. For cDNA copy numbers higher than 240, the APC secreation rate per cDNA and per cell decreased dramatically. The γ-carboxylation of glutamic acid residues, a posttranslational modification required for APC biological activity, was also investigated. The proportion of APC that was fully γ-carboxylated decreased as the secretion rate of APC increased. © 1995 John Wiley & Sons, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260460104
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  • 5
    Electronic Resource
    Electronic Resource
    Targeted antisense modulation of inflammatory cytokine receptors (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 72-81 
    Details
    ISSN: 0006-3592
    Keywords: antisense ; receptor targeted delivery ; acute phase response ; IL-6 ; gp 130 ; HepG2 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Antisense technology is potentially a powerful means by which to selectively control gene expression. We have used antisense oligonucleotides to modulate the response of the hepatoma cell line, HepG2, to the inflammatory cytokine, IL-6, by inhibiting the expression of its multifunctional signal transducer, gp130. HepG2 cells respond to IL-6 by upregulating acute phase proteins, such as haptoglobin, by five- to tenfold. Gp130 is central to this response, as the upregulation of haptoglobin is almost completely blocked by the addition of high concentrations (∼100 μg/ml) of a monoclonal antibody to gp 130. Antisense oligodeoxynucleotides complementary to the mRNA encoding gp 130 inhibited the upregulation of haptoglobin by IL-6-stimulated HepG2 cells by about 50%. However, a nonsense sequence also inhibited haptoglobin secretion by about 20%. To improve the specificity and efficiency of action, we targeted the antisense oligonucleotides to HepG2 cells using a conjugate of asialoglycoprotein-poly-L-lysine. The targeted antisense reduced the binding of IL-6 to HepG2 cells, virtually eliminating high affinity binding. In addition, it inhibited haptoglobin upregulation by over 70%. Furthermore, the dose of targeted antisense required for biological effect was reduced by about an order of magnitude as compared with unconjugated antisense. These results demonstrate the potential of antisense oligonucleotides as a means to control the acute phase response as well as the need for a greater understanding of the mechanism and dynamics of antisense molecules as they are developed toward therapeutic application. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 72-81, 1997.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Kinetic modeling of poly(β-hydroxybutyrate) production and consumption by Paracoccus pantotrophus under dynamic substrate supply (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 773-782 
    Details
    ISSN: 0006-3592
    Keywords: PHB ; poly(β-hydroxybutyrate) ; Paracoccus pantotrophus ; dynamic growth ; metabolic modeling ; polymers ; activated sludge process ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of the research was to obtain insights into the behavior of microorganisms under feast/famine conditions as often occur in wastewater treatment processes. The response of microorganisms to such conditions is the accumulation of storage polymers like poly(β-hydroxybutyrate). The research was performed using a pure culture of Paracoccus pantotrophus LMD 94.21. A steady-state C-limited chemostat culture was switched to batch mode and a pulse of acetate was added. As long as external substrate (acetic acid) was present, the organism grew and accumulated poly(β-hydroxybutyrate). After depletion of the external substrate, the stored poly(β-hydroxybutyrate) was used as growth substrate. Poly(β-hydroxybutyrate) accumulation was found to be strongly dependent on the growth rate of the organism before the pulse addition of acetate. Poly(β-hydroxybutyrate) accumulation was correlated to the difference in maximum acetate uptake rate and the acetate required for growth. Based on the interpretation of the experimental results, a metabolically structured model has been set up. This model adequately describes the observed kinetics of the poly(β-hydroxybutyrate) formation and consumption. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 773-782, 1997.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Nitrite inhibition of denitrification by Pseudomonas fluorescens (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 194-201 
    Details
    ISSN: 0006-3592
    Keywords: denitrification kinetics ; nitrite inhibition model ; Pseudomonas fluorescens ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studies. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred-tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 μg N/L. Denitrification activity was partially associated with growth, as described by the Luedeking-Piret equation. However, when the freshly inoculated reactor was operated discontinuosly, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuoulsy exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (grown on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch. © 1995 John Wiley & Sons, Inc.
    Additional Material: 12 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260460303
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  • 8
    Electronic Resource
    Electronic Resource
    Deactivation and conformational changes of cutinase in reverse micelles (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 380-386 
    Details
    ISSN: 0006-3592
    Keywords: reverse micelles ; cutinase ; deactivation ; conformational changes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380-386, 1998.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Application of empirical design methodologies to the study of the influence of reaction conditions and N-α-protecting group structure on the enzymatic X-Phe-Leu-NH2 dipeptide synthesis in buffer/dimethylformamide solvents systems (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 539-549 
    Details
    ISSN: 0006-3592
    Keywords: enzymatic peptide synthesis ; N-terminal protecting groups ; α-chymotrypsin ; experimental design ; partition constant ; reaction rate ; log P ; molecular refractivity ; response surfaces ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the α-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH2 was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-α protection choice. Boc and Z were the most reactive N-α groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260390509
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  • 10
    Electronic Resource
    Electronic Resource
    Blood plasma levels of lipoperoxides, glutathione peroxidase, beta carotene, vitamin A and E in women with habitual abortion (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Cell Biochemistry and Function 16 (1998), S. 227-231 
    Details
    ISSN: 0263-6484
    Keywords: lipid peroxidation ; antioxidants ; habitual abortion ; vitamin A and E ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The plasma levels of lipoperoxides, glutathione peroxidase (GSH-Px), reduced glutathione (GSH), beta carotene, vitamin A, E, some plasma biochemical and blood haematological parameters were investigated in 40 women with habitual abortion (HA) and controls. The levels of GSH, vitamin A, E and beta carotene were significantly lower in women with HA than in controls. However, the plasma levels of lipid peroxidation, alkaline phosphatase (ALP), glucose and blood haemoglobin were significantly higher in HA than in controls. In addition, plasma levels of GSH-Px, AST, ALT, total bilirubin, total protein, albumin, sodium, potassium, calcium and number of white blood cells, red blood cells, platelet and values of packet cell volume showed no significant differences between HA and controls. According to the results of this study, we observed that the levels of lipid peroxidation were increased and plasma levels of vitamin A, E and beta carotene were decreased in HA. The decrease of those antioxidants may play a significant role in women with habitual abortion. Copyright © 1998 John Wiley & Sons, Ltd.
    Additional Material: 3 Tab.
    Type of Medium: Electronic Resource
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