bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monitoring glutamine in animal cell cultures using a chemiluminescence fiber optic biosensor
Authors: Cattaneo M. V. , Luong J. H. T. 
Published: December 2004

AbstractTogether with flow injection analysis (FIA), a chemiluminescence (CL) fiber optic biosensor system has been developed for determining glutamine in animal cell cultures. Glutaminase (GAH) and glutamate oxidase (GLO) were onto separate porous aminopropyl glass beads via glutaraldehyde activation and packed to form an enzyme column. These two enzymes acted in sequence on glutamine to produce hydrogen peroxide, which was then reacted with luminol in the presence of ferricyanide to produce a light signal. An anion exchanger was introduced on‐line to eliminate interfering endogenous glutamate in view of its negative charge at pH above 3.22 (isoelectric pH). Among several resins tested, the acetate form was most effective, and this type of ion exchanger also effectively adsorbed uric acid, acetaminophen, and aspartic acid.There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 1 to 100 μM. A complete analysis could be performed in 2 min, including sampling and washing with a good reproducibility (± 4.4%). Both the bi‐enzymic and ion exchange columns were useful for at least 500 analyses when the biosensor system was applied for the glutamine determination in murine hybridoma cell cultures and insect cell cultures. The values obtained compared well with those of HPLC, thus validating the applicability of the CL fiber optic system. © 1993 John Wiley & Sons, Inc.
A comparison of oxygenation methods fro high‐density perfusion culture of animal cells
Authors: Zhang S. , Handa‐Corrigan A. , Spier R. E. 
Published: December 2004

AbstractA perfusion culture system was developed to investigate the oxygenation of high‐density hybridoma cell cultures. The culture system was composed of a stirred‐tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble (≈5 mm in diameter), micron‐sized bubble (≈ 80 μm in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron‐sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble‐free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high‐density animal cell cultures, provided that the culture medium is supplemented with Pluronic F‐68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long‐term cultivation were found to be more problematic than simple bubble oxygenation of high‐density cell culture. The micron‐sized bubble oxygenation method is highly recommended for high‐density animal cell cultures, provided that Pluronic F‐68 is supplemented into the culture medium. © 1993 John Wiley & Sons, Inc.
A structured model describing carbon and phosphate limited growth of Catharanthus roseus plant cell suspensions in batch and chemostat culture
Authors: van Gulik W. M. , ten Hoopen H. J. G. , Heijnen J. J. 
Published: December 2004

AbstractThe growth of plant cell suspension cultures of Catharanthus roseus in batch fermentors was studied at different initial phosphate levels of the medium. On the basis of the observations and existing knowledge with respect to phosphate metabolism in cultured C. roseus cells, a structured mathematical model was developed for the description of the kinetics of growth and intracellular accumulation of glucose and phosphate, as a function of glucose and phosphate supply. It was shown that the model offers not only good description of the growth of the cells in batch culture at different initial phosphate levels, but also provided a satisfactory description of the growth in glucose limited chemostats. © 1993 Wiley & Sons, Inc.
Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima
Authors: Brown Stephen H. , Sjøholm Carsten , Kelly Robert M. 
Published: December 2004

AbstractThermotoga maritima, among the most thermophilic eubacteria currently known, produces glucose isomerase when grow in the presence of xylose. The purified enzyme is a homotetramer with submit molecular Wight of about 45,000. It has a number of features in common with previously described glucose isomerases‐pH optimum of 6.5 to 7.5, presence of activesite histidine, requirement for metal cations such as Co2+ and Mg2+, and preference for xylose as substrate. In addition, it has significant sequence/structural homology with other glucose isomerases, as shown by both N‐terminal sequencing and immunological crossreactivity. The T. maritima enzyme is distinguished by its extreme thermostability–a temperature optimum of 105 to 110°C, and an estimated half‐life of 10 minutes at 120°C, pH 7.0. The high degree of thermostability, coupled with a neutral to slightly acid pH optimum, reveal this enzyme to be a promising candidate for improvement of the industrial glucose isomerization process © 1993 Wiley & Sons, Inc.
Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p‐xylene by two Pseudomonas isolates.
Authors: Chang Myung‐Keun , Voice Thomas C. , Criddle Craig S. 
Published: December 2004

AbstractTwo Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot‐scale fluidized bed reactor treating groundwater containing benzene, toluene, and p‐xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p‐xylene in the presence of toluene. Strain X1 grew on toluene and p‐xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth‐rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p‐xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p‐xylene. © 1993 Wiley & Sons, Inc.
Effect of fluorochrome location in protein A on sensing efficiency of IgG
Authors: Huang Ping Yu , Lee Cheng S. 
Published: December 2004

AbstractFragment B of protein A conjugated with fluorescein at various lysine residues is prepared and separated by using a DEAE column in anion‐exchange chromatography. The binding of IgG Fc to fragment B contributes to an additional positive electric potential around fragment B. The change in the local electrostatic environment and pH can then be specifically monitored by measuring the fluorescence intensity of fluorescein conjugated with fragment B before and after the introduction of IgG. The studies for the quantitative dependence of fluorescein location on the effectiveness of fluorescein for sensing the protein A–IgG reaction are presented and discussed. © 1993 John Wiley & Sons, Inc.
Regeneration of NADH in a bioreactor using yeast cells immobilized in alginate fiber: I. Method and effect of reactor variables
Authors: Stevenson E. , Ibbotson P. G. , Spedding P. L. 
Published: December 2004

AbstractSaccharomyces cerevisiae cells immobilized in a calcium alginate fiber reactor were used as a source of alcohol dehydrogenase for the NAD+‐to‐NADH reaction. The reaction was catalyzed by enzyme in cells on the surface of the fiber. Internal diffusional effects were present. The enzyme cell concentration was optimized by harvesting cells finally grown under anaerobic conditions. The results were expressed as an apparent reaction rate constant that was independent of NAD+ and excess ethanol concentration, was slightly affected by flow rate above a minimum value, and increased with immobilized cell concentration in the fiber. The reaction was complete after 6 to 7 h under optimal conditions of 36°C and 9.5 pH. The latter was 0.5 pH units above the free enzyme optimum, indicating that microenvironmental effects were in evidence. © 1993 John Wiley & Sons, Inc.
Construction, expression, and analysis of recombinant HIV gp41 constructs containing a novel cellular binding domain
Authors: Kestler D. P. , Henderson L. A. , Noti J. D. 
Published: December 2004

AbstractThe gp41 polypeptide of human immunodeficiency virus (HIV) contains an immunosuppressive domain, an epitope which elicits specific cytolytic T cell responses to HIV, and a complement Clq interactive domain. In addition, a synthetic peptide called CS3, derived from gp41 (amino acids 576–593 of gp160) and contiguous with the major immunodominant domain, binds to cellular proteins and may be important in HIV entry/fusion. In order to further investigate the role of the CS3 region of gp41 in cellular binding and to investigate other properties of gp41, sufficient quantities of this polypeptide must be readily available. We have therefore cloned the region of the HIV genome between nucleotides 7891 and 8188 (corresponding to amino acids 541–639 of gp160) into a series of procaryotic expression vectors. The resulting clones express a recombinant polypeptide of gp41 (r41). Two of these recombinants, pMAL‐cRl/r41 and pGEMEX‐2/r41, expressed the highest and most consistent levels of r41 as judged by both sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis. With the pMAL‐cRl/r41 construct, r41 was expressed as a fusion to the maltose‐binding protein (MBP) and, following purification by affinity chromatography, was cleaved from MBP by factor Xa protease digestion. MBP/r41 may be useful for studies of a reported gp41 cellular binding domain and may facilitate studies involving other functions ascribed to this region of gp41. © 1993 John Wiley & Sons, Inc.
Esterification reactions catalyzed by lipases in microemulsions: The role of enzyme localization in relation to its selectivity
Authors: Stamatis H. , Xenakis A. , Provelegiou M. , Kolisis F. N. 
Published: December 2004

AbstractThe activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis‐(2‐ethylhexyl)sulfo‐succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies. © 1993 John Wiley & Sons, Inc.
Transport of 1‐μm latex particles in pseudomonas aeruginosa biofilms
Authors: Drury William J. , Stewart Philip S. , Characklis William G. 
Published: December 2004

AbstractFluorescent latex microbeads added to a Pseudomonas aeruginosa biofilm as tracers of particle movement penetrated the biofilm and remained in it much longer than predicted by a model of advective displacement due to cell growth. Beads with a nominal diameter of 1 μm that were added in the bulk fluid became distributed throughout the biofilm depth. Some microbeads penetrated to the substratum within the 24‐h bead addition period. The biofilms had a mean thickness of approximately 34 μm but have been previously shown to be quite rough. Measured rates of bead release from the biofilm corresponded to first order time coefficients of 0.01–0.03 h−1. These bead release rates were approximately an order of magnitude less than the predicted time scale of advective transport, which is just the experimentally measured specific cellular growth rate of 0.15 h−1. Computer simulations of bead transport using the biofilm model BIOSIM were compared with bead release rate data and with bead position distributions within the biofilm as determined by microscopic examination of thin cross sections of embedded biofilm. The model predicted much faster release of beads from the biofilm than actually occurred. It is hypothesized that both the ability of beads to penetrate the biofilm and the unexpectedly low advective displacement velocity of particles in the biofilm were due to the rough nature of the biofilm. © 1993 John Wiley & Sons, Inc.
A three‐phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture
Authors: Bushell M. E. , Bell S. L. , Scott M. F. , Snell K. , Spier R. E. , Wardell J. N. , Sanders P. G. 
Published: December 2004

AbstractThe use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using 15NH4Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis‐appearance of mRNA consistent with the three phases indicated by the fermentation parameters. © 1993 John Wiley & Sons, Inc.
A synergistic kinetics model for enzymatic cellulose hydrolysis compared to degree‐of‐synergism experimental results
Authors: Converse A. O. , Optekar J. D. 
Published: December 2004

AbstractIt is demonstrated that a two‐enzyme component synergistic model can account for the observation that the degree of synergism goes through a maximum as the total enzyme concentration is increased. The degree of synergism is low at low enzyme concentration because the extent of conversion is low and therefore the cellulose chain ends, present originally, are not exhausted; thus the action of the cellobiohydrolase (CBH) is not dependent on the chain ends generated by the endoglucanase (EG). The degree of synergism declines at high enzyme concentration due to saturation of adsorption sites with CBH, thus decreasing the generation of chain ends by EG. © 1993 John Wiley & Sons, Inc.
Production of recombinant proteins in high‐density insect cell cultures
Authors: Reuveny S. , Kim Y. J. , Kemp C. W. , Shiloach J. 
Published: December 2004

AbstractThe effect of the growth phase of Spodoptera frugiperda (Sf9) cells on the production of recombinant proteins (β‐galactosidase and glucocerebrosidase) was investigated. Cells infected with the recombinant Autographa californica nuclear polyhedrosis virus at the late exponential and stationary phases yielded low quantities of expressed protein. Highest enzyme yields were obtained using Sf9 cells from the early exponential phase (0.9 mg β‐galactosidase/106 cells and 1.7 μg glucocerebrosidase/106 cells). Infection of resuspension of cells collected from various phases of growth in fresh medium resulted in 75% restoration of maximal expression levels. This finding suggested either nutrient limitation or waste product accumulation as the cause of the decrease in productivity at the latter phases of growth. Further experiments revealed that the highest productivity levels could be obtained with cultures of Sf9 cells grown in a fermentor to a cell concentration of 4 × 106 mL−1. The medium needed to be replaced prior to infection with the recombinant virus and supplemented with a mixture of glucose, L‐glutamine, and yeastolate ultrafiltrate. © 1993 John Wiley & Sons, Inc.
A bioassay of thyrotrophin by photografted mammalian cells onto polymeric supports
Authors: D'Ambrosio Andrea , Bellobono Ignazio Renato , Marangoni Francesca , Rebulla Paolo , Sirchia Girolamo 
Published: December 2004

AbstractViable and functionally responsive human thyroid follicular cells, suspended in a commercial polyester acrylate diluted with tripropyleneglycol diacrylate, photoinitiated, and photocatalyzed with a proprietary photocatalytic system based on a synergic mixture of vanadium (V) t‐butoxide and i‐propoxide, have been immobilized as monolayers onto polystyrene plates. Bioassay of thyrotrophin in immobilized cell cultures yielded, by log‐log plot of the dose–response curve, a slope (0.92 ± 0.02) in close agreement with that (0.91) reported for cells immobilized by physical adsorption. The decisive role of photocatalyst in the photografting procedure has also been shown experimentally. A mechanism is suggested by which cells are anchored, rapidly and with stable chemical bonds, onto one of the two acrylate functions of the monomer–prepolymer mixture, the other one being simultaneously responsible for photochemical grafting onto the support. © 1993 John Wiley & Sons, Inc.
The cellulose‐binding domain (CBDCex) of an exoglucanase from Cellulomonas fimi: Production in Escherichia coli and characterization of the polypeptide
Authors: Ong Edgar , Gilkes Neil R. , Miller Robert C. , Warren R. Antony J. , Kilburn Douglas G. 
Published: December 2004

AbstractThe gene fragment encoding the cellulose‐binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi was subcloned and expressed in Escherichia coli. Transcription from the lac promoter coupled with translation from a consensus prokaryotic ribosome binding site led to the production of large quantities of CBDCex (up to 25% total soluble cell protein). The polypeptide leaked into the culture supernatant (up to 50 mg · L−1), facilitating one‐step purification by affinity chromatography on cellulose. The 11‐kDa polypeptide reacted with Cex antiserum. Absence of free thiols indicated that the two Cys residues of CBDCex form a disulfide bridge. It had the same N‐terminal amino acid sequence as CBDCex prepared from Cex by proteolysis, plus two additional N‐terminal amino acid residues (Ala and Ser) encoded by the Nhel site introduced during plasmid construction. CBDCex bound to a variety of β‐1, 4‐glycans with different affinities and saturation levels. Adsorption to bacterial microcrystalline cellulose was dependent on the temperature, but not on the pH. © 1993 John Wiley & Sons, Inc.
Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila
Authors: Guisán José M. , Alvaro Gregorio , Fernandez‐Lafuente Roberto , Rosell Cristina M. , Garcia Jose L. , Tagliani Auro 
Published: December 2004

AbstractWe have developed a strategy for immobilization–stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose–aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme–support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000‐fold more stable than soluble and one‐point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)]. © 1993 John Wiley & Sons, Inc.
Lipase catalyzed esterification of glycidol in organic solvents
Authors: Martins João F. , Da Ponte Manuel Nunes , Barreiros Susana 
Published: December 2004

AbstractWe studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n‐octanol–water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35°C. The enzyme exhibited maximum activity at a water content of 13 ± 2% (w/w). The enantiomeric purity obtained was 83 ± 2% of (S)‐glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. © 1993 John Wiley & Sons, Inc.