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  • Biochemistry and Biotechnology  (13,095)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 590-599 
    ISSN: 0006-3592
    Keywords: protein refolding ; hollow-fibre membrane ; dialysis ; carbonic anhydrase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration membrane to refold bovine carbonic anhydrase, loaded into the lumen space, by removing the denaturant through controlled dialysis via the shell side space. When challenged with GdnHCl-denatured carbonic anhydrase, 70% of the loaded protein reptated through the membrane into the circulating dialysis buffer. Reptation occurred because the protein, in its fully unfolded configuration, was able to pass through the pores. The loss of carbonic anhydrase through the membrane was controlled by the dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min reduced the denaturant around the protein to a concentration that allowed the return of secondary structure, increasing the hydrodynamic radius, thus preventing protein transmission. Under these conditions a maximum of 42% of carbonic anhydrase was recovered (from a starting concentration of 5 mg/mL) with 94% activity. This is an improvement over refolding carbonic anhydrase by simple batch dilution, which gave a maximum reactivation of 85% with 35% soluble protein yield. The batch refolding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25°C the temperature sensitivity was considerably reduced. In order to reduce the convection forces that give rise to aggregation and promote refolding the dialyzate was slowly heated from 4 to 25°C. This slow, temperature-controlled refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membrane, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 590-599, 1998.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 119-120 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 658-662 
    ISSN: 0006-3592
    Keywords: T4 lysozyme ; silica nanoparticles ; synthetic enzyme variants ; surface-induced conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Maintaining a specific molecular conformation is essential for the proper functioning of an enzyme. A substantial loss of catalytic activity can occur from the displacement caused by even a single amino acid substitution. Activity may also be lost as an enzyme undergoes a conformational change during adsorption. In this study, we investigated the effect of thermostability on the activities of three T4 lysozyme variants after adsorption to 9 nm colloidal silica particles. Less-stable T4 lysozyme variants lost more activity after adsorption than did more stable variants, apparently because they experienced more extensive structural alteration. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 658-662, 1998.
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  • 4
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 139-148 
    ISSN: 0006-3592
    Keywords: metabolic engineering ; pathway analysis ; metabolic and energetic model ; physiological state ; Saccharomyces cerevisiae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this work, an integrated modeling approach based on a metabolic signal flow diagram and cellular energetics was used to model the metabolic pathway analysis for the cultivation of yeast on glucose. This approach enables us to make a clear analysis of the flow direction of the carbon fluxes in the metabolic pathways as well as of the degree of activation of a particular pathway for the synthesis of biomaterials for cell growth. The analyses demonstrate that the main metabolic pathways of Saccharomyces cerevisiae change significantly during batch culture. Carbon flow direction is toward glycolysis to satisfy the increase of requirement for precursors and energy. The enzymatic activation of TCA cycle seems to always be at normal level, which may result in the overflow of ethanol due to its limited capacity. The advantage of this approach is that it adopts both virtues of the metabolic signal flow diagram and the simple network analysis method, focusing on the investigation of the flow directions of carbon fluxes and the degree of activation of a particular pathway or reaction loop. All of the variables used in the model equations were determined on-line; the information obtained from the calculated metabolic coefficients may result in a better understanding of cell physiology and help to evaluate the state of the cell culture process. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:139-148, 1998.
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  • 5
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 149-153 
    ISSN: 0006-3592
    Keywords: Metabolic Control Analysis ; flux control coefficients ; top down MCA ; metabolic engineering ; Corynebacterium glutamicum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Grouping of reactions around key metabolite branch points can facilitate the study of metabolic control of complex metabolic networks. This top-down Metabolic Control Analysis is exemplified through the introduction of group (flux, as well as concentration) control coefficients whose magnitudes provide a measure of the relative impact of each reaction group on the overall network flux, as well as on the overall network stability, following enzymatic amplification. In this article, we demonstrate the application of previously developed theory to the determination of group flux control coefficients. Experimental data for the changes in metabolic fluxes obtained in response to the introduction of six different environmental perturbations are used to determine the group flux control coefficients for three reaction groups formed around the phosphoenolpyruvate/pyruvate branch point. The consistency of the obtained group flux control coefficient estimates is systematically analyzed to ensure that all necessary conditions are satisfied. The magnitudes of the determined control coefficients suggest that the control of lysine production flux in Corynebacterium glutamicum cells at a growth base state resides within the lysine biosynthetic pathway that begins with the PEP/PYR carboxylation anaplorotic pathway. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:149-153, 1998.
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  • 6
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 154-161 
    ISSN: 0006-3592
    Keywords: central carbon pathways ; metabolic optimization ; ethanol production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Many attempts to engineer cellular metabolism have failed due to the complexity of cellular functions. Mathematical and computational methods are needed that can organize the available experimental information, and provide insight and guidance for successful metabolic engineering. Two such methods are reviewed here. Both methods employ a (log)linear kinetic model of metabolism that is constructed based on enzyme kinetics characteristics. The first method allows the description of the dynamic responses of metabolic systems subject to spatiotemporal variations in their parameters. The second method considers the product-oriented, constrained optimization of metabolic reaction networks using mixed-integer linear programming methods. The optimization framework is used in order to identify the combinations of the metabolic characteristics of the glycolytic enzymes from yeast and bacteria that will maximize ethanol production. The methods are also applied to the design of microbial ethanol production metabolism. The results of the calculations are in qualitative agreement with experimental data presented here. Experiments and calculations suggest that, in resting Escherichia coli cells, ethanol production and glucose uptake rates can be increased by 30% and 20%, respectively, by overexpression of a deregulated pyruvate kinase, while increase in phosphofructokinase expression levels has no effect on ethanol production and glucose uptake rates. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:154-161, 1998.
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  • 7
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 170-174 
    ISSN: 0006-3592
    Keywords: catabolite repression ; phosphotransferase system ; inducer exclusion ; inducer expulsion ; protein kinase ; transcriptional regulation ; transport regulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Catabolite repression is a universal phenomenon, found in virtually all living organisms. These organisms range from the simplest bacteria to higher fungi, plants, and animals. A mechanism involving cyclic AMP and its receptor protein (CRP) in Escherichia coli was established years ago, and this mechanism has been assumed by many to serve as the prototype for catabolite repression in all organisms. However, recent studies have shown that this mechanism is restricted to enteric bacteria and their close relatives. Cyclic AMP-independent mechanisms of catabolite repression occur in other bacteria, yeast, plants, and even E. coli. In fact, single-celled organisms such as E. coli, Bacillus subtilis, and Saccharomyces cerevisiae exhibit multiple mechanisms of catabolite repression, and most of these are cyclic AMP-independent. The mechanistic features of the best of such characterized processes are briefly reviewed, and references are provided that will allow the reader to delve more deeply into these subjects. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:170-174, 1998.
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  • 8
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 162-169 
    ISSN: 0006-3592
    Keywords: bioinformatics ; metabolic engineering ; genetic engineering ; mathematical analysis ; stoichiometry ; enzyme kinetics ; modal analysis ; genetic circuits ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:162-169, 1998.
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  • 9
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 191-195 
    ISSN: 0006-3592
    Keywords: control analysis ; Lactococcus lactis ; gene expression ; flux ; oligonucleotide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts. In a recent approach, artificial promoters for modulating gene expression in micro-organisms were constructed using synthetic degenerated oligonucleotides. From this work, a promoter library was obtained for Lactococcus lactis, containing numerous individual promoters and covering a wide range of promoter activities. Importantly, the range of promoter activities was covered in small steps of activity change. Promoter libraries generated by this approach allow for optimization of gene expression and for experimental control analysis in a wide range of biological systems by choosing from the promoter library promoters giving, e.g., 25%, 50%, 200%, and 400% of the normal expression level of the gene in question. If the relevant variable (e.g., the flux or yield) is then measured with each of these constructs, then one can calculate the control coefficient and determine the optimal expression level. One advantage of the method is that the construct which is found to have the optimal expression level is then, in principle, ready for use in the industrial fermentation process; another advantage is that the system can be used to optimize the expression of different enzymes within the same cell. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:191-195, 1998.
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  • 10
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 175-190 
    ISSN: 0006-3592
    Keywords: protein-based polymers ; inverse temperature transitions ; hydrophobic-induced pKa shifts ; waters of hydrophobic hydration ; five axioms for protein engineering; microwave dielectric relaxation ; a universal mechanism for biological energy conversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill.This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions.Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity.Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:175-190, 1998.
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  • 11
    ISSN: 0006-3592
    Keywords: Escherichia coli ; Chloramphenicol Acetyltransferase (CAT) ; Culture Redox Potential (CRP) ; Dithiothreitol (DTT) ; reducing agents ; molecular chaperones ; proteases ; heat shock ; stress response ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/μg total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/μg CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (σ32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/μg CAT min.) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 248-259, 1998.
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  • 12
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    Biotechnology and Bioengineering 59 (1998), S. 261-272 
    ISSN: 0006-3592
    Keywords: effective diffusive permeability ; diffusion coefficient ; biofilm ; cell density ; review ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes ( 〉 5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:261-272, 1998.
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  • 13
    Electronic Resource
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    Biotechnology and Bioengineering 59 (1998), S. 281-285 
    ISSN: 0006-3592
    Keywords: protein aggregation ; RNase A ; protein formulation ; protein additives ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the previous study (part I), heat-denatured RNase A aggregation was shown to depend on the solution pH. Interestingly, at pH 3.0, the protein did not aggregate even when exposed to 75°C for 24 h. In this study, electrostatic repulsion was shown to be responsible for the absence of aggregates at that pH. While RNase A aggregation was prevented at the extremely acidic pH, this is not an environment conducive to maintaining protein function in general. Therefore, attempts were made to confer electrostatic repulsion near neutral pH. In this study, heat-denatured RNase A was mixed with charged polymers at pH 7.8 in an attempt to provide the protein with excess surface cations or anions. At 75°C, SDS and dextran sulfate were successful in preventing RNase A aggregation, whereas their cationic, nonionic, and zwitterionic analogs did not do so. We believe that the SO3- groups present in both additives transformed the protein into polyanionic species, and this may have provided a sufficient level of electrostatic repulsion at pH 7.8 and 75°C to prevent aggregation from proceeding. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:281-285, 1998.
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  • 14
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    Biotechnology and Bioengineering 59 (1998), S. 328-343 
    ISSN: 0006-3592
    Keywords: biotrickling filters ; biotrickling filter modeling ; mono-chlorobenzene ; biodegradation kinetics of mono-chlorobenzene ; chlorinated VOC emissions ; biofiltration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Removal of mono-chlorobenzene (m-CB) vapor from airstreams was studied in a biotrickling filter (BTF) operating under counter-current flow of the air and liquid streams. Experiments were performed under various values of inlet m-CB concentration, air and/or liquid volumetric flow rates, and pH of the recirculating liquid. Conversion of m-CB was never below 70% and at low concentrations exceeded 90%. A maximum removal rate of about 60 gm-3-reactor h-1 was observed. Conversion of m-CB was found to increase as the values of liquid and air flow rate increase and decrease, respectively. The effects of pH and frequency of medium replenishment on BTF performance were also investigated. The process was successfully described with a detailed mathematical model, which accounts for mass transfer and kinetic effects based on m-CB and oxygen availability. Solution of the model equations yielded m-CB and oxygen concentration profiles in all three phases (airstream, liquid, biofilm). It is predicted that oxygen has a controling effect on the process at high inlet m-CB concentrations. From independent, suspended culture, experiments it was found that m-CB biodegradation follows Andrews inhibitory kinetics. The kinetic constants were found to remain practically unchanged after the culture was used in BTF experiments for 8 months. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:328-343, 1998.
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  • 15
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    Biotechnology and Bioengineering 59 (1998), S. 344-350 
    ISSN: 0006-3592
    Keywords: electrodialysis ; citric acid ; pH ; temperature ; Faraday efficiency ; solute recovery efficiency ; specific energy consumption ; solute flux ; water flux ; feed solute concentration ; electric current density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of pH and temperature (θ) on the overall performance indicators (i.e., solute recovery, ρ, and Faraday, η, efficiencies; specific energy consumption, ε, solute, JS, and water, JW, fluxes) of batch electrodialytic recovery of citric acid from model solutions was assessed at different values of feed solute concentration (cSf) and electric current density (j). Regardless of the initial feed concentration used, ρ and JS were found to be independent of θ; η and JW exhibited a positive trend with respect to θ, while ε a negative one. At the maximum temperature tested (33°C), as the pH of the feed solution was varied from 3 to 7, ρ increased from 0.90 ± 0.08 to 0.97 ± 0.02, η grew from 0.09 ± 0.02 to 0.50 ± 0.01, JS practically doubled, ε reduced about 8 times, but JW increased from 3 to 4 times. So, the optimal conditions for this technique are to be determined by balancing the savings in the investment and maintenance costs against the energy costs. © John Wiley & Sons, Inc. Biotechnol Bioeng 59:344-350, 1998.
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  • 16
    ISSN: 0006-3592
    Keywords: chymotrypsin ; enzyme stability ; reversed micelles ; interface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stability of α-chymotrypsin and δ-chymotrypsin was studied in reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. α-Chymotrypsin is inactivated at the interface and at the water pool, while δ-chymotrypsin is inactivated only at the water pool. The mechanism of inactivation at the interface is related to the interaction of N-terminal group alanine 149 (absent in δ-chymotrypsin) with the negative interface. The dependence of enzyme activity on water content of these two enzymes in reversed micelles of AOT is also related with the interface interaction, since δ-chymotrypsin does not have a bell-shaped curve as observed for α-chymotrypsin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:360-363, 1998.
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  • 17
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    Biotechnology and Bioengineering 59 (1998), S. 351-359 
    ISSN: 0006-3592
    Keywords: bioreactor ; high density ; insect cells ; perfusion ; Sf9 ; ultrasonic filter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The baculovirus/insect cell expression system has provided a vital tool to produce a high level of active proteins for many applications. We have developed a very high-density insect cell perfusion process with an ultrasonic filter as a cell retention device. The separation efficiency of the filter was studied under various operating conditions. A cell density of over 30 million cells/mL was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. Sf9 cells from a high-density culture and a spinner culture were infected with two recombinant baculoviruses expressing genes for the production of human chitinase and monocyte-colony inhibition factor. The protein yield on a cell basis from infecting high-density Sf9 cells was the same as or higher than that from the spinner Sf9 culture. Virus production from the high-density culture was similar to that from the spinner culture. The results show that the ultrasonic filter did not affect insect cells' ability to support protein expression and virus production following infection with baculovirus. The potential applications of the high-density perfusion culture for large-scale protein expression from Sf9 cells are also highlighted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:351-359, 1998.
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  • 18
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    Biotechnology and Bioengineering 59 (1998), S. 374-378 
    ISSN: 0006-3592
    Keywords: conductive paint electrode ; prevention of marine biofouling ; fishing net ; alternating potential ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conductive paint electrode was used for marine biofouling on fishing nets by electrochemical disinfection. When a potential of 1.2 V vs. a saturated calomel electrode (SCE) was applied to the conductive paint electrode, Vibrio alginolyticus cells attached on the electrode were completely killed. By applying a negative potential, the attached cells were removed from the surface of the electrode. Changes in pH and chlorine concentration were not observed at potentials in the range -0.6 ∼1.2 V vs. SCE. In a field experiment, accumulation of the bacterial cells and formation of biofilms on the electrode were prevented by application of an alternating potential, and 94% of attachment of the biofouling organisms was inhibited electrically on yarn used for fishing net coated with conductive paint. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:374-378, 1998.
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  • 19
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    Biotechnology and Bioengineering 59 (1998), S. 364-373 
    ISSN: 0006-3592
    Keywords: porous supports ; internal and external diffusion ; active site accessibility ; enzyme loading ; kinetically controlled dipeptide synthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mass transfer limitations were studied in enzyme preparations of α-chymotrypsin made by deposition on different porous support materials such as controlled pore glasses, Celite, and polyamides of different particle sizes. It is the onset of mass transfer limitations that determines the position of the activity optimum with respect to enzyme loading on each support. The evidence of various experiments indicates that internal diffusional limitations are the important mechanism for the observed mass transfer limitations. External diffusion was not found to play an important role under the conditions used, and it was also found that when immobilizing multilayers of enzyme the buried enzyme molecules are active to a large extent. An extreme situation is observed on Celite at very high loadings. Under these conditions, this support is expected to have its pores completely filled with packed enzyme molecules, and then it is the diffusion within the enzyme layer that determines the observed rate. As the enzyme loading increases, the area of contact between the deposited enzyme layers and the liquid solution inside the pores diminishes, causing a decrease on the observed rate of an intrinsically fast reaction which apparently is incongruous with the presence of more enzyme in the system. This work shows that mass transfer limitations can be an important factor when working with immobilized enzymes in organic media, and its study should be carried out in order to avoid undesired reduced enzyme activities and specificities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:364-373, 1998.
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  • 20
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    Biotechnology and Bioengineering 59 (1998), S. 438-444 
    ISSN: 0006-3592
    Keywords: bioremediation ; plasma discharge ; dichlorophenol degradation ; perchloroethylene degradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pulsed electric discharge (PED) and bioremediation were combined to create a novel two-stage system which dechlorinates the halogenated pollutants, 2,4-dichlorophenol and perchloroethylene, with repetitive (0.1-1 kHz), short pulse (∼100 ns), low voltage (40-80 kV) discharges and then mineralizes the less chlorinated products with aerobic bacteria. A 6.1 mM aqueous dichlorophenol sample was cycled through the PED reactor (60 kV of applied pulsed voltage and 300 Hz) 6 times, resulting in the release of 55% of the initial dichlorophenol chloride ions (1 mM Cl- removed each cycle). The respective average specific efficiency is 0.4-0.6 keV/(Cl- molecule). Pseudomonas mendocina KR1, which grows in minimal medium supplemented with phenol but not with dichlorophenol, increased in cell density in all cultures supplemented with the PED-treated DCP samples and yielded a maximum of two-fold additional Cl- released compared to the PED-related alone. The number of PED-treatment cycles, voltage, and frequency were also varied, showing that both cell densities and overall dichlorophenol dechlorination were highly dependent upon the number of PED-treatment cycles, rather than the tested voltages and frequencies. Using this two-stage treatment system, PED released 31% of the initial chloride ions from dichlorophenol (after three cycles at 40-45 kV and 1.2 kHz) while P. mendocina KR1 in the second stage increased dechlorination to 90%. These results were corroborated by the 35% additional chloride release found with activated sludge cultures. Perchloroethylene (0.6 mM) was similarly treated in a first-stage PED reactor (80% chloride removal after four cycles) followed by biodegradation of the dechlorinated products with a recombinant toluene o-monooxygenase-expressing Pseudomonas fluorescens strain. Gas chromatographic analysis showed that the PED reactor created less-chlorinated byproducts (i.e., trichloroethylene) that were removed (74%) upon exposure to the recombinant bacterium. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:438-444, 1998.
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  • 21
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    Biotechnology and Bioengineering 59 (1998), S. 445-450 
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosylation engineering ; antisense ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:445-450, 1998.
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  • 22
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    Biotechnology and Bioengineering 59 (1998), S. 451-460 
    ISSN: 0006-3592
    Keywords: protein fouling ; membrane transport ; ultrafiltration ; adsorption ; filtration ; composite membrane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:451-460, 1998.
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  • 23
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    Biotechnology and Bioengineering 59 (1998), S. 461-470 
    ISSN: 0006-3592
    Keywords: aqueous two-phase separation ; protein partitioning ; T4 lysozyme ; electrochemical partitioning ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components.We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:461-470, 1998.
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  • 24
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    Biotechnology and Bioengineering 57 (1998), S. 518-528 
    ISSN: 0006-3592
    Keywords: ammonium ; UDP-GlcNAc ; N -glycosylation ; BHK-21 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of different ammonium concentrations and glucosamine on baby hamster kidney (BHK)-21 cell cultures grown in continuously perfused double membrane bioreactors was investigated with respect to the final carbohydrate structures of a secretory recombinant glycoprotein. The human interleukin-2 (IL-2) mutant glycoprotein variant IL-Mu6, which bears a novel N-glycosylation site (created by a single amino acid exchange of Gln100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH4Cl, or glucosamine. Recombinant glycoprotein products were purified and characterized by amino acid sequencing and carbohydrate structural analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry, high-pH anion-exchange chromatography with pulsed amperometric detection, and methylation analysis. In the absence of glutamine, cells secreted glycoprotein forms with preponderantly biantennary, proximal fucosylated carbohydrate chains (85%) with a higher NeuAc content (58%). Under standard conditions in the presence of 7.5 mM glutamine, complex-type N-glycans were found to be mainly biantennary (68%) and triantennary structures (33%) with about 50% containing proximal α1-6-linked fucose; 37% of the antenna were found to be substituted with terminal α2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH4Cl, a significant and reproducible increase in tri- and tetraantennary oligosaccharides (45% of total) was detected in the secretion product. In glutamin-free cultures supplemented with glucosamine, an intermediate amount of high antennary glycans was detected. The increase in complexity of N-linked oligosaccharides is considered to be brought about by the increased levels of intracellular uridine diphosphate-GlcNAc/GalNAc. These nucleotide sugar pools were found to be significantly elevated in the presence of high NH3/NH4+ and glucosamine concentrations. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 518-528, 1998.
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  • 25
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    Biotechnology and Bioengineering 57 (1998), S. 557-570 
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; polyhydroxyalkanoates ; metabolic engineering ; mathematical modeling ; enzyme kinetics ; regulation of metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 557-570, 1998.
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  • 26
    ISSN: 0006-3592
    Keywords: rhG-CSF ; fusion protein ; secretion efficiency ; glycosylation ; multimer ; conformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GAL1-10 upstream activating sequence (UASG), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1β. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1β fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O-glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 600-609, 1998.
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  • 27
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    Biotechnology and Bioengineering 57 (1998), S. 620-623 
    ISSN: 0006-3592
    Keywords: protein refolding ; reversed micelles ; solid-liquid extraction ; RNase A ; DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 620-623, 1998.
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  • 28
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    Biotechnology and Bioengineering 57 (1998), S. 610-619 
    ISSN: 0006-3592
    Keywords: dynamic model ; Saccharomyces cerevisiae ; oxidative capacity ; feedback control ; calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this study was to characterize the dynamic adaptation of the oxidative capacity of Saccharomyces cerevisiae to an increase in the glucose supply rate and its implications for the control of a continuous culture designed to produce biomass without allowing glucose to be diverted into the reductive metabolism. Continuous cultures subjected to a sudden shift-up in the dilution rate showed that the glucose uptake rate increased immediately to the new feeding rate but that the oxygen consumption could not follow fast enough to ensure a completely oxidative metabolism. Thus, part of the glucose assimilated was degraded by the reductive metabolism, resulting in a temporary decrease of biomass concentration, even if the final dilution rate was below Dcrit. The dynamic increase of the specific oxygen consumption rate, qO2, was characterized by an initial immediate jump followed by a first-order increase to the maximum value. It could be modeled using three parameters denoted qjumpO2, qmaxO2, and a time constant τ. The values for the first two of the parameters varied considerably from one shift to another, even when they were performed under identical conditions. On the basis of this model, a time-dependent feed flow rate function was derived that should permit an increase in the dilution rate from one value to another without provoking the appearance of reductive metabolism. The idea was to increase the glucose supply in parallel with the dynamic increase of the oxidative capacity of the culture, so that all of the assimilated glucose could always be oxidized. Nevertheless, corresponding feed-profile experiments showed that deviations in the reductive metabolism could not be completely suppressed due to variability in the model parameters. Therefore, a proportional feedback controller using heat evolution rate measurements was implemented. Calorimetry provides an excellent and rapid estimate of the metabolic activity. Satisfactory control was achieved and led to constant biomass yields. Ethanol accumulated only up to 0.49 g L-1 as compared to an accumulation of 1.82 g L-1 without on-line control in the shift-up experiment to the same final dilution rate. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 610-619, 1998.
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  • 29
    ISSN: 0006-3592
    Keywords: c-jun ; cell cycle ; apoptosis ; antisense ; growth deprivation ; F-MEL ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: F-MEL cells were transfected with the c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and the obtained cells were named c-jun AS cells. When the c-jun AS cells were treated with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth of the cells was completely suppressed for a duration of 16 days with a high cell viability exceeding 86%. The c-jun expression in the c-jun AS cells was suppressed moderately in the absence of DEX and strongly in the presence of DEX. The c-jun AS cells grew well and reached a density of 106 cells/mL without supplementation of any serum components. Viability was greater than 80% after the cells had been cultured for 8 days in the absence of DEX. The c-jun AS cells stayed at a constant cell density and high viability above 80% for 8 days when they were cultured in the presence of DEX under serum deprivation. In contrast, the wild type F-MEL cells were unable to grow and died by apoptosis in 3 days under serum deprivation. Internucleosomal cleavage of DNA, a landmark of apoptosis, was clearly detectable. Thus the c-jun AS cell line that is resistant to apoptosis induced by serum deprivation and can reversibly and viably be growth-arrested was established. A dual-signal model was proposed to explain the experimental result, the interlinked regulation of apoptosis, and growth by c-jun.© 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:65-72, 1998.
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  • 30
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    Biotechnology and Bioengineering 58 (1998), S. 380-386 
    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.
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  • 31
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 63-68 
    ISSN: 0884-3996
    Keywords: ethanol ; hexachlorobenzene ; porphyria ; oxidative stress ; spontaneous urinary chemiluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Hexachlorobenzene (HCB) administration to rats induces porphyria cutanea tarda, characterized by high levels of urinary porphyrins (〉40 μg/day) and accumulation of highly carboxylated porphyrins in liver (〉15 μg/g of tissue). Ethanol administration, under the conditions employed, was not porphyrinogenic and was able to diminish some of the responses elicited by HCB. Furthermore, ethanol and/or HCB administration leads to organ disturbances that involve oxidative stress. We have measured the changes in urinary chemiluminescence (CL) levels, as part of a systematic evaluation of the metabolic alterations in rats chronically treated with ethanol and/or HCB. The results, that constitute the first set of urinary CL data obtained from an animal model system, indicate that the measurement of the spontaneous urinary CL can constitute a fast, simple and sensitive method to evaluate disturbances associated with oxidative stress. © 1998 John Wiley & Sons, Ltd.
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  • 32
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 85-90 
    ISSN: 0884-3996
    Keywords: stopped-flow ; chemiluminescence ; multicomponent analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The stopped-flow technique was employed to measure chemiluminescent emission from the reaction of a mixture of oxalate and proline with a chemiluminescence reagent, tris(2,2′-bipyridine)ruthenium(III), or Ru(bpy)33+. Ru(bpy)33+ is a versatile reagent and is often used in bioanalytical applications, including the detection of certain drugs and their metabolites, for example. Unfortunately, Ru(bpy)33+ has not yet been fully examined as a possible chemiluminescence reagent for simultaneous kinetic determinations. In this work, a differential reaction rate method, based on simple least squares regressions of the pseudo-first order decay data, was used to resolve two compounds, oxalate and proline, reacting simultaneously with Ru(bpy)33+. Our results indicate that stopped-flow analyses with Ru(bpy)33+ could provide a viable method for simultaneous determinations of unresolvable analytes of environmental and pharmaceutical importance. © 1998 John Wiley & Sons, Ltd.
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  • 33
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 287-293 
    ISSN: 0884-3996
    Keywords: electroporation ; UV light ; oxidative stress ; catalase ; superoxide dismutase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Escherichia coli JM101 cells were subjected to pore-forming electric fields, irradiation with ultraviolet light or oxidative stress by either the lipoxygenase products 9- and 13-hydroperoxyoctadecadienoic acids (9- and 13-HPOD) or hydrogen peroxide. It was found that all chemico-physical stresses enhanced ultraweak light emission from the bacterial cells, the most effective treatment being electroporation (up to 20-fold increase in luminescence compared to the control value), followed by oxidative stress with 9- or 13-HPOD (up to 4-fold increase) and irradiation with UV light (up to 2.8-fold increase). Bacterial luminescence was always in the red edge of the spectrum and was paralleled by changes in membrane oxidative index and specific activity of catalase and superoxide dismutase. © 1998 John Wiley & Sons, Ltd.
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  • 34
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 307-309 
    ISSN: 0884-3996
    Keywords: immunoassay ; biological markers ; myocardial infarction ; diagnostic sensitivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Three new immunochemiluminometric assays for quantitation of cardiac markers, i.e. creatine kinase isoenzyme MB (CK-MB), myoglobin and cardiac troponin I (cTnI), were evaluated with the Sanofi Access analyser. The complete profile requires 20 min to perform, the method being suitable in true stat situations. In patients with early myocardial infarction (median time of sample collection: 210 min from onset, range 30-450; n = 44), the diagnostic sensitivity of Access cTnI was 66%, compared with 80% for myoglobin, and 43% for CK-MB. For comparison, cTnI, with an automated immunofluorimetric assay was also measured (sensitivity, 45%; p 〈 0.05 vs. Access cTnI). Our data confirmed myoglobin as the first biochemical marker to appear elevated after infarction. However, cTnI may be a more sensitive marker for early detection of cardiac damage than initially thought, when determined by an ultrasensitive method such as an immunochemiluminometric assay. © 1998 John Wiley & Sons, Ltd.
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  • 35
    ISSN: 0884-3996
    Keywords: bioluminescence ; adrenalin ; noradrenalin ; photophores ; HPLC ; mesopelagic fish ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The presence of adrenalin (E) and noradrenalin (NE) was found by HPLC both in the photophores and at other tissue levels of numerous species of mesopelagic fish in The Strait of Messina, with the aim of determining the incidence of these catecholamines in photophores, in light transmission and the eventual presence at other tissue levels. © 1998 John Wiley & Sons, Ltd.
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  • 36
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 273-278 
    ISSN: 0884-3996
    Keywords: diabetes ; leukocytes ; monocytes ; extracellular matrix ; non-enzymatic glycosylation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Non-enzymatic glycosylation (NEG) of collagen has been previously shown to significantly influence the reactive oxygen metabolism (ROM) of phagocytic cells in healthy subjects. Considering the role of NEG in the pathophysiology of diabetes, we have further analysed the oxidative metabolism of polymorphonuclear cells (PMNs) and monocytes in 23 patients with non-insulin dependent diabetes mellitus in order to better elucidate a possible pathogenic role of NEG of the extracellular matrix in long-term complications of diabetes. Experiments were performed in triplicate on native-collagen and glycated-collagen coated vials, using a chemiluminescence (CL) assay. Results show that PMNs from diabetic patients display a significant increased basal and zymosan-induced CL activity with respect to controls that are not related to the glycation state of the substrate. Conversely, the CL activity of monocytes induced by zymosan shows a decrease in diabetic patients with respect to healthy volunteers (p 〈 0.05). Moreover, monocyte CL was reduced by the glycated matrix, both in healthy volunteers and in diabetic subjects (p 〈 0.05 and p 〈 0.01, respectively). These data highlight a complex role of phagocytic leukocytes in the pathophysiology of extracellular matrix alterations secondary to NEG that are typically present in clinical conditions such as diabetes or ageing. © 1998 John Wiley & Sons, Ltd.
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  • 37
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 349-354 
    ISSN: 0884-3996
    Keywords: β-D-galactosidase ; enzyme immunoassay ; chemiluminescence ; 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) ; thyroxine ; indole derivative ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We developed a novel chemiluminescent assay of β-D-galactosidase (β-gal) based on the chemiluminescence of indole. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was used as a substrate for β-gal and also as a light emitter. X-gal was hydrolysed by β-gal to liberate free indoxyl, followed by oxidation to indigo dye, and simultaneously produces hydrogen peroxide (H2O2). H2O2 reacts with the residual X-gal in the presence of horseradish peroxidase (HRP) to emit light. The measurable range of β-gal obtained by this method was 6 × 10-14 mol/L to 6 × 10-11 mol/L; the detection limit was 3 amol/assay. This chemiluminescent assay could be applied to an enzyme immunoassay of thyroxine using β-gal as the enzyme label. © 1998 John Wiley & Sons, Ltd.
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  • 38
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 21-24 
    ISSN: 0884-3996
    Keywords: 2-D imaging ; chemiluminescence ; auto-oxidation ; hydration ; cereal products ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional (2-D) spatial distributions of ultra-weak chemiluminescence (photon imaging) from auto-oxidizing and water-hydrated cereal food products were measured by means of a high-sensitivity 2-D photon counting system - an intensified charge coupled device (CCD) camera. The 2-D images obtained reveal the dynamics and emission patterns of very slow auto-oxidation reactions and much faster processes of water penetration into cereal products. The enhancement of chemiluminescence by the addition of water appears to involve complex processes with an inhomogenous spatial and energy distribution within cereal products. The effect of antioxidants, free radical promoters and scavengers suggests that oxidative radical reactions contribute significantly to the observed chemiluminescence. The possible involvement of hydrophilic interactions, H2O-biopolymers and recombination of trapped radicals is also discussed. © 1998 John Wiley & Sons, Ltd.
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  • 39
    ISSN: 0884-3996
    Keywords: CRF ; TAC ; lipids ; apolipoproteins ; oxLDLAb ; diet ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Lipoprotein abnormalities are common in uraemia and are considered important factors for development of atherosclerosis and progression of renal disease. Reduction of total antioxidant capacity (TAC) and lipid peroxidation (LP) probably play a major role in both processes. The aim of this study was to assess the effect of renal function, dietary manipulation and lipids on TAC of uraemic patients with different chronic renal failure (CRF). Sixty patients (36M, 24F), aged 60 ± 12 years were divided into five groups according to serum creatinine levels (sCr, mg/dl) -  CRFI, 1.5-3; CRFII, 〉 3-5.5; CRFIII, 〉 5.5; CRFIV, 〉 3 on vegetarian supplemented diet (SD); CRFV haemodialysis patients (HD)-and investigated for TAC by enhanced chemiluminescent assay, autoantibodies against oxidized LDL (oxLDLAb), lipids, apolipoprotein AI, B, Lp(a) and uric acid (UA). The results were compared to a control group of 19 people (8M, 11F), aged 52 ± 11 years with sCr 〈 1.5. TAC increased significantly with the progression of CRF and was strongly related to both sCr and UA. Lipids and SD did not show any influence on TAC. Unexpectedly, lipid peroxidation did not correlate to TAC, neither to sCr or UA. HD accounted for a mild reduction of both TAC and LP. Patients on SD showed a marked reduction of LP as compared to patients with a similar degree of renal failure (CRF-III) but on conventional diet. Our results suggest that elevated TAC in uraemia is likely to be dependent on increased UA levels and does not seem to induce an effective protection in vivo from oxidative stress. In conclusion, TAC does not appear to be a reliable method for assessing the oxidative susceptibility of CRF patients. © 1998 John Wiley & Sons, Ltd.
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  • 40
    ISSN: 0884-3996
    Keywords: chemiluminescence ; PCR ; contamination ; polymorphism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Minisatellite analysis is commonly used in forensic disputes but can also be applied to the investigation of cell contamination. Such a problem arises, for example, when transplantation is performed. The presence of contamination has been investigated by other authors using radioactive methods. In the present study we describe a method that allows the detection of contamination with high sensitivity without using radioactive substances. Our technique is based on the use of polymerase chain reaction (PCR) amplification of minisatellite sequences (VNTR), followed by chemiluminescent detection. In particular, biotin-labelled dCTP is included in the PCR mixture and detection of PCR products is obtained following the CSPD chemiluminescent protocol (Southern-Light Nucleic Acid Detection Systems). We applied this method to artificial mixes of DNA of two individuals with alleles of different sizes. We performed progressive dilutions of an individual DNA into the other's DNA and revealed a contamination of 1 in 2500 cells. We also tested our technique searching for maternal contamination in cord blood samples in 60 cases and revealed a 18.3% contamination. The technique that we set up proves to be a very sensitive one which could be applied not only to the detection of maternal cells in cord blood but also in studying any other kind of contamination. © 1998 John Wiley & Sons, Ltd.
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  • 41
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 295-301 
    ISSN: 0884-3996
    Keywords: plant virus ; diagnosis ; transgenic plants ; non-radioactive probes ; digoxigenin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Due to costs in using and disposing of radiochemicals and to health considerations, we have been developing applications which include non-isotopic detection of DNA and proteins using chemiluminescence. Our major interests are in the detection of viral nucleic acids and in the analysis of transgenic plants. Generally, probes were labelled with digoxigenin, either by the random priming method or by PCR, and then detected with CSPD or CDP-Star. We routinely use a tissue blotting protocol for diagnosing TYLCV, a plant virus becoming a pest in the Mediterranean region. Test results were comparable with those using the same radiolabelled probe. When total nucleic acids are extracted from the plant samples and used in dot-blot or Southern blot assays, viral DNAs are promptly detected by chemiluminescence. In transgenic plants, chemiluminescence was used to detect the transgene on genomic Southern blots, the transgenic mRNAs on Northern blots, and the transgenic protein on Western blots. In Southern and Northern blots, the quality of the results obtained was usually satisfactory, but not as good as with a radiolabelled probe, the main problem being the signal-to-background ratio. Our goal is now to improve the quality of results in demanding applications such as genomic Southern blots, by reducing the background on membranes. © 1998 John Wiley & Sons, Ltd.
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  • 42
    ISSN: 0884-3996
    Keywords: chemiluminescence ; β-galactosidase ; tetracyclines ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The observation that tetracyclines inhibit the biosynthesis of β-galactosidase in Escherichia coli to a greater extent than other antibacterials was exploited for the development of a chemiluminometric method to detect residues of this class of antibiotics in milk. The procedure involves the incubation of a milk sample with 107 CFU/ml of an E. coli strain in the presence of IPTG, an inducer of β-galactosidase, and of EGTA, a chelator of calcium ions, followed by a 1000-fold dilution and measurement of the residual enzymatic activity using the chemiluminogenic substrate Galacton. Chemiluminometry proved an essential tool in this procedure because the extensive dilution of the sample, necessary to avoid light quenching by turbidity, results in an insufficient level of β-galactosidase activity to be measurable by colorimetry. This tetracycline galactosidase (TG) test has been validated and compared in the field to existing commercial screening assays for antibiotics. Its detection limit for tetracyclines ranges between 40 and 65 μg/kg, which is below the European maximum residue limit (MRL = 100 μg/kg) in milk. No other antibacterials, at concentrations commonly expected in milk, were found to interfere with the TG test. Strategies to avoid false positive reactions possibly arising from very high somatic cell counts will be reported elsewhere. © 1998 John Wiley & Sons, Ltd.
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  • 43
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 355-363 
    ISSN: 0884-3996
    Keywords: hydrogen peroxide ; sodium hypochlorite ; reactive oxygen species ; chemiluminescence ; luminol ; lucigenin ; penicillin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H2O2 is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe2+ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co2+ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co2+. Cu2+ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration-reaction curve is not as steep. NaOCl enhances H2O2/Fe2+-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 104 (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity. © 1998 John Wiley & Sons, Ltd.
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  • 44
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 371-378 
    ISSN: 0884-3996
    Keywords: bioluminescence ; luciferase ; ATP ; immobilization ; glass ; poly-L-lysine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The bioluminescent reaction catalysed by firefly luciferase has become widely established as an outstanding analytical system for assay of ATP. When used in solution, luciferase is unstable and cannot be re-used, a problem that can be partially circumvented by immobilizing the enzyme on solid substrates. Transparent glass is especially advantageous over alternative immobilizing matrices, since it allows most of the emitted photons to be detected. We report a new method for luciferase immobilization on glass which does not require prior silanization and glutaraldehyde activation, thus saving preparation time and minimizing enzyme inactivation. Our method is based on the co-immobilization by adsorption of luciferase (from a firefly lantern extract) and poly-L-lysine (PL) on non-porous glass strips. Luciferase immobilized in this way exhibits minimal variations in intersample activity, high sensitivity for ATP detection (linear luminescence responses down to 50 nmol/L) and good stability (full activity for at least 60 days when stored at -80°C). PL-mediated immobilization of luciferase on glass strips provides an attractive strategy for the design of specific ATP biosensors, with potential in industry, environmental screening, medicine and biological research. © 1998 John Wiley & Sons, Ltd.
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  • 45
    ISSN: 0884-3996
    Keywords: bile acids ; chemiluminescence ; superoxide ; antioxidants ; micelles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe2+-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.
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  • 46
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 365-369 
    ISSN: 0884-3996
    Keywords: Vibrio fischeri ; LuxR ; lux ; bioluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We have recently suggested that the expression of V. fischeri right lux operon is initiated from two sites, the first located upstream of the luxI gene, while the second seems to be located upstream of the luxC gene. The transcription from both sites is negatively controlled by H-NS protein. E. coli MC4100 rpoS hns mutant harbouring the V. fischeri luxCDABE genes showed constitutive mode and 70,000-fold higher luminescence than the wild-type cells. The present study shows that the expression of luxCDABE genes in E. coli MC4100 wild-type cells is also controlled by LuxR protein in the absence of the autoinducer. The co-presence of a ptac-controlled luxR gene in a trans position to a plasmid carrying the luxCDABE genes resulted in 100,000 times higher luminescence. In the absence of the autoinducer, the presence of the luxR gene under its own regulated control resulted in about 100-200-fold increase of luminescence from the luxC upstream site. Taken together, it seems that the LuxR protein initiates the formation of the V. fischeri lux system cloned in E. coli from two sites located upstream and downstream of the luxI gene. Only the activation of the first site requires the presence of the autoinducer, whereas the second site is fully activated by LuxR protein in the absence of the autoinducer. © 1998 John Wiley & Sons, Ltd.
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  • 47
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: No Abstract
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  • 48
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 139-145 
    ISSN: 0884-3996
    Keywords: electrochemiluminescence ; metal ions ; 2,3-diaminonaphthalene ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Electrochemiluminescence (ECL) of 200 ppm 2,3-diaminonaphthalene (2,3-DAN) was studied alone and in conjunction with 100 ppm of 34 different metal and non-metal ions and revealed three relatively intense ECL responses from interactions of 2,3-DAN with Au+, Fe+3 and V+5. ECL responses from Cr+6 or Ru+3 with 2,3-DAN were less intense, but noteworthy, as was the coloured fluorescent product of the non-metal ion Se+4 interaction with 2,3-DAN. Several intense 2,3-DAN-metal ion ECL reactions were studied in greater detail and revealed various titration curves with ionic detection limits in the low ppm range, using a fixed level (200 ppm) of 2,3-DAN. © 1998 John Wiley & Sons, Ltd.
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  • 49
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 185-188 
    ISSN: 0884-3996
    Keywords: Vibrio fischeri ; H-NS ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We have recently proposed that the expression of V. fischeri right lux operon is controlled by two promoters; the first one located upstream of the luxI gene, while the second one seems to be located upstream of the luxC gene. The transcription from both promoters is negatively controlled by H-NS protein. Escherichia coli MC4100 rpoS hns mutant that carried the V. fischeri lux system with a deletion in either the luxI or luxR gene showed a constitutive mode and more than 10,000-fold higher luminescence than the control cells. The present study shows that neither luxR nor luxI are required for the transcription of the luxCDABE genes in an H-NS deficient strain of E. coli. The MC4100 rpoS hns mutant harbouring the luxCDABE-carrying plasmid showed constitutive mode and 70,000-fold higher luminescence than the wild-type cells. The question whether both the left and the right operons of V. fischeri lux system are controlled by H-NS was addressed with the aid of plasmids harbouring the lacZ gene fused with luxR or luxI. In MC4100 hns rpoS background, luxR and luxI genes were very early and actively transcribed, as judged by the strong β-galactosidase activity that was developed at early stage of growth. The β-galactosidase activity in the wild-type cells was 20-40 times lower and occurred mainly during the second half of the growth cycle. It thus appears that H-NS inhibits the transcription of three promoters of the lux system of V. fischeri; the left operon that codes for LuxR protein and two promoters located upstream and downstream to luxI gene. © 1998 John Wiley & Sons, Ltd.
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  • 50
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    Journal of Bioluminescence and Chemiluminescence 13 (1998), S. 285-285 
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: No abstract.
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  • 51
    ISSN: 0884-3996
    Keywords: free radicals ; antioxidant ; total antioxidant capacity ; Trolox ; Insulin-dependent diabetes mellitus (IDDM) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Free radicals are considered to be important factors involved in many physiopathological processes. Several methods have been proposed for studying the mechanisms of antioxidant protection against free radical-induced injury, including the measurement of the total antioxidant capacity (TAC) in body fluids, based on enhanced chemiluminescence. This technique is calibrated against Trolox™ and assay results are expressed as μmol/L of Trolox equivalents. Since many of the complications induced by diabetes appear to be mediated by oxygen free radical generation, we have investigated serum antioxidant capacity in a group of healthy subjects and in insulin-dependent diabetic (IDDM) subjects. A statistically significant difference was noticed in TAC values between the IDDM group and the young control group. Even if the biological meaning of this significant reduction in TAC remains to be explained, an overproduction of precursors of reactive oxygen free radicals and/or a decreased scavenger systems efficiency can be associated with the increased risk of atherosclerotic cardiovascular disease in diabetic patients. © 1998 John Wiley & Sons, Ltd.
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  • 52
    ISSN: 0884-3996
    Keywords: peroxynitrite ; antioxidants ; luminol ; acetaminophen ; phenols ; catecholamines ; norepinephrine ; isoproterenol ; epinephrine ; SIN-1 ; sydnonimines ; polyphenols ; catechins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: This study is based on a simple chemical interaction of peroxynitrite (O=N—O—O-) and luminol, which produces blue light upon oxidation. Since peroxynitrite has a half-life of about 1 s, a drug known as linsidomine (SIN-1) is used as a peroxynitrite generator. Peroxynitrite can oxidize lipids, proteins and nucleic acids. Upon the stimulation of inflammation and/or infection, macrophages and neutrophils can be induced to produce large amounts of peroxynitrite, which can oxidize phenols and sulphhydryl-containing compounds. Therefore, phenols and sulphhydryls eliminate peroxynitrite. This is an example of the Yin-Yang hypothesis e.g. oxidation-reduction. Acetaminophen (Tylenol®) can inhibit fever and some types of pain without being a particularly effective anti-inflammatory. Since it is a phenol, it could act as a nitration target for peroxynitrite. Then peroxynitrite, the possible cause of pain and elevated temperature, might be destroyed in the reaction. Acetaminophen is a phenolic compound which produces a clear inhibitory dose-response curve with peroxynitrite in its range of clinical effectiveness. Whether acetaminophen actually works as we suggest is to be proven. Three different types of reaction could decrease the amount of peroxynitrite: (a) interference with base-catalysed opening of the SIN-1 molecule; (b) destruction of one or both substances needed to form it -  superoxide and/or nitric oxide; when the SIN-1 degrades to superoxide and nitric oxide, the former may be destroyed by superoxide dismutase (SOD); (c) peroxynitrite may react directly with phenols (mono-, di-, tri- and tetraphenols), possibly by nitration. Nordihydroguaiaretic acid and 2-hydroxyestradiol (catechol estrogen) are potent inhibitors of luminol light emission. Epineprine, isoproterenol, pyrogallol, catechol and ascorbic acid (a classic antioxidant) are all inhibitors of luminol chemiluminescence. Isoproterenol, norepinephrine/and epinephrine first inhibit light but overall stimulate the light production. Initially, SIN-1 degrades to produce peroxynitrite, which reacts with luminol to produce blue light. If any of three catecholamines are present with the reaction that produces light, there is an initial inhibition of light production, and then a marked stimulation. A possible reason for this is that these catechols are oxidized and the metabolized phenol stimulates the production of light from luminol. Also, during oxidation of catecholamines superoxide is sometimes formed, which could stimulate production of peroxynitrite. This simple screening system is introduced to find useful antioxidants against peroxynitrite. © 1998 John Wiley & Sons, Ltd.
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  • 53
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    Biotechnology and Bioengineering 57 (1998), S. 46-54 
    ISSN: 0006-3592
    Keywords: smooth muscle ; polyglycolic acid ; biodegradable ; tissue engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 × 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 ± 0.8 × 108 cells/cm3 after 5 weeks, compared to 2.0 ± 1.1 × 108 cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 ± 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 46-54, 1998.
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  • 54
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    Biotechnology and Bioengineering 57 (1998), S. 87-94 
    ISSN: 0006-3592
    Keywords: flow cytometry ; spectrofluorymetry ; intracellular protein and nucleic acids quantification ; cell viability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of flow cytometry (FCM) to quantitatively analyze intracellular compounds is studied. FCM is a very useful technique for individual cell studies in microbial systems, and gives access to information which cannot be obtained in any other way. Nevertheless, it provides data in arbitrary units, that is, relative data. This analytical technique could be employed for kinetic modeling of microbial systems and even for internal phenomena analysis, but for this purpose, absolute data - that is concentration of intracellular compounds - must be used.In this work, relative flow cytometry data are transformed into absolute data by means of calibrations employing the same fluorochromes with another technique: spectrofluorymetry. Calibrations of DNA, RNA, and protein intracellular concentrations are presented for the bacteria, Xanthomonas campestris. Other analytical methods, based on biochemical determinations, were also employed to quantify intracellular compounds, but the results obtained are very poor compared with those achieved by means of spectrofluorymetry (SFM). Calibration equations and data obtained by both techniques are given. Evolutions of protein and nucleic acids during Xanthomonas campestris growth and xanthan gum production are shown. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 87-94, 1998.
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  • 55
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    Biotechnology and Bioengineering 57 (1998), S. 109-117 
    ISSN: 0006-3592
    Keywords: nanofiltration ; selectivity ; inorganic membrane ; peptide ; pH ; ionic strength ; polyelectrolyte ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nanofiltration (NF) membrane technology shows interesting potentials for separating organic components on the basis of solute charge and size in the range of 300-1000 g mol-1. Separation properties of two inorganic NF membranes were studied with a set of 10 small peptides (molecular mass range: 300-900 g mol-1; 3 〈 pI 〈 10) contained in a well-characterized tryptic β casein hydrolysate. Peptides transmission strongly depended on ionic interactions in the system. Physicochemical conditions such as ionic strength and especially pH were crucial to the separation, because the membrane and peptides showed amphoteric properties. Thus, the three categories of peptides (acid, basic, neutral) were separated according to their pI because of presumed concentration gradients of charged peptides at the membrane: positive for basic peptides and negative for acid peptides. At optimum pH 8 this led to high transmissions of basic peptides (even over 100%), intermediate transmissions for neutral peptides, and low transmissions for acid peptides. The addition of multicharged cationic and anionic species in the hydrolysate induced a markedly enhanced selectivity when the polyelectrolyte was a membrane coion and a complete reversion of selectivity when it was a membrane counterion. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 109-117, 1988.
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  • 56
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    Biotechnology and Bioengineering 57 (1998), S. 127-135 
    ISSN: 0006-3592
    Keywords: membrane mass spectrometer ; kinetic measurements ; anaerobic biofilm ; acetate ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A small, stirred, 14.4-mL tank reactor was designed to serve as a measurement cell for short-term investigation of microbial kinetics. A mass spectrometer membrane probe allowed the measurement of the dissolved gases of hydrogen, methane, oxygen, and carbon dioxide. pH was measured by an electrode and controlled by addition of acid or alkali. The highly sensitive measurement of gases with low solubility allowed rapid measurements at very low conversion. In kinetic experiments, a stepwise increase of substrate concentration (method A) and continuous feed of substrate (method B) were used, allowing quick estimation of substrate kinetics. Acetate conversion in mixed culture biofilms from a fluidized bed reactor was investigated. Substrate inhibition was found to be negligible in the concentration range studied. Experiments at various pH values showed that the undissociated acid form was the kinetic determinant. Kinetic parameters for Haldane kinetics of protons were KSH = 1.3 × 10-5 mol m-3 and KIH = 8.1 × 10-3 mol m-3. With free acid (HAc) as the rate determining species, the kinetic parameters for method A were KSHAc = 0.005 mol m-3 and KIHAc = 100 mol m-3 and for method B were KSHAc = 0.2 mol m-3 and KIHAc = 50 mol m-3. The maximum biomass activity occurred at around pH 6.5. Acetate was exclusively converted to methane and CO2 at pH 〉 6. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 127-135, 1998.
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