bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Situ Fluorescence Cell Mass Measurements of Saccharomyces cerevisiae Using Cellular Tryptophan
Authors: Horvath John J. , Glazier Scott A. , Spangler Christopher J. 
Published: March 2005

AbstractThis work describes a new spectroscopic optical fiber/rod technique for in situ real time measurement of cell mass and product concentrations in bioreactors using intrinsic fluorescence. The variable excitation/emission wavelength capability of this sensor allows for species‐selective measurement during fermentations. Cell mass (tryptophan) and product concentrations (pyridoxine) have been measured during fermentations of Saccharomyces cerevisiae. The effects of varying substrate concentration and oxygen concentration on the observed cell mass signals are eliminated by direct measurement of cell mass, as opposed to indirect measurement schemes such as those using NADH fluorescence. The sensor is robust and able to undergo many cycles of in situ steam sterilization without degradation, and its fluorescence signal is linear with concentration for all species studied in this work. Tryptophan fluorescence from yeast is shown to be a better measure of cell mass than NADH fluorescence.
Amino Acid Overproduction and Catabolic Pathway Regulation in Saccharomyces cerevisiae
Authors: Martinez‐Force Enrique , Benítez Tahia 
Published: March 2005

AbstractTo determine whether blocking a degradative pathway leads to amino acid accumulation, the internal free concentrations of threonine, methionine, and related amino acids have been measured in a strain of Saccharomyces cerevisiae and its mutants lacking either the L‐threoninedeaminase enzyme (EC 4.2.1.16) codedfor by the ILV1 gene (ilv mutants), that coded for by the CHA1 gene (cha− mutants), or both threonine deaminase enzymes (ilv−cha− mutants). Whereas maximal accumulation of internal free amino acids occurred in the double mutants ilv−cha−, ilv− single mutants displayed amino acid concentrations higher than those of either the wild type or the cha− single mutant. On the other hand, when these enzymes were measured in threonine and methionine overproducer mutants of an industrial strain of Saccharomyces cerevisiae, results indicated, in most cases, lower activities than those of the wild type, although there was not a total correlation between maximal threonine and/or methionine accumulation and minimal enzymatic activities. Results point to the isolation of strains that accumulate essential amino acids by blocking their degradative pathway.
Cultivation of Recombinant Baby Hamster Kidney Cells in a Fluidized Bed Bioreactor System with Porous Borosilicate Glass
Authors: Kratje Ricardo B. , Reimann Antje , Hammer Joachim , Wagner Roland 
Published: March 2005

AbstractDense cell cultivation of the recombinant cell line BHK 21 pSVIL2 was performed in a fluidized bed bioreactor system containing porous borosilicate glass carriers. Experiments were carried out with different medium formulations for a period of 48 days. Due to an effective immobilization of the cells in the reactor, continuous operation was easy to perform. Maximal cell densities and product yields could be maintained, even when protein‐free medium was perfused exceeding 2 reactor volumes per day. Final cell densities of magnitude 7.1 ± 107 mL−1 intrasphere volume were reached, while the interleukin‐2 production rate was 0.70 mg day−1. The cell specific productivity reached a value of 1.3 ± 10−10 mg day−1. The first results were presented with a cell line that grows under glutamine‐f ree medium conditions. The use of a glutamine‐f ree medium for the cultivation of the cells resulted in a drastic decrease in cell metabolism. Furthermore, the amino acids lysine and histidine were produced and secreted into the culture supernatant, although these metabolites normally are considered to be essential for animal cells grown in vitro. However, no lethal effect on the cells has been detected, and the total number of cells in the reactor remained constant. The metabolism of threonine has been detected to be directly dependent on the presence of glutamine. Cells grown in glutamine‐free culture medium produced glycine yields 6 times higher than those grown in glutamine‐containing medium. A bead‐to‐bead transfer of the cells has also been detected when the cells immobilized within the intrasphere volume of the borosilicate carriers reached the stationary phase.
Technology for Regenerable Biosensor Probes Based on Enzyme‐Cellulose Binding Domain Conjugates
Authors: Phelps Michael R. , Hobbs John B. , Kilburn Douglas G. , Turner Robin F. B. 
Published: March 2005

AbstractThe application of enzyme‐based biosensors for on‐line bioprocess monitoring and control has been slowed by problems relating to the in situ sterilizability of the probe and the stability of the enzyme component. A novel technology with the potential to address both of these difficulties is presented here. The approach is based on the reversible immobilization of enzymes conjugated with the cellulose binding domain (CBD) of cellulases from Cellulomonas fimi. A regenerable biosensor electrode can be configured with a cellulose matrix onto which the enzyme‐CBD conjugate can be repeatedly loaded (bound by the CBD) and subsequently eluted by perfusing the cellulose matrix with the appropriate solution. Glucose oxidase (GOx) conjugated to CBD with glutaraldehyde was used in an experimental glucose biosensor to demonstrate the feasibility of multiple cycles of loading and elution of the conjugate. Michaelis‐Menten enzyme kinetics provided an empirical model for the calibration of the experimental biosensor. The development of a computer‐controlled prototype glucose biosensor and a fermentation monitoring system based on this approach is discussed.
Characteristics and Performance of an Autotuning Proportional Integral Derivative Controller for Dissolved Oxygen Concentration
Authors: Lee Seung Chul , Hwang Young Bo , Lee Tae Ho , Chang Yong Keun , Chang Ho Nam 
Published: March 2005

AbstractThe characteristics and performance of an autotuning proportional integral derivative (PID) controller developed by us have been investigated. The parameters for this controller, proportional gain, K, the integral time constant, Ti, and the derivative time constant, Td, are on‐line‐tuned by using heuristic rules, requiring no α priori tuning. The controller showed a stable and robust performance, even when only K was on‐line‐tuned. However, the dynamic (or time delay) of the membrane‐type dissolved oxygen (DO) probe caused oscillations in the DO concentration unless it was properly compensated. This required a prediction method. When all three controller parameters were on‐line‐adjusted, the DO probe dynamic could be effectively overcome with no separate prediction step.
A Hybrid Centrifuge Rotor for Continuous Bioprocessing
Authors: Ivory Cornelius F. , Gilmartin Michael , Gobie William A. , Mcdonald Craig A. , Zollars Richard L. 
Published: March 2005

AbstractAll existing centrifuges have a resolving power that is proportional to their centrifugal field, G = ω2R, raised to the first power. By combining conventional sedimentation with field‐flow fractionation in a two‐dimensional separation system, it is possible to devise a continuous rotor whose selectivity is proportional to the second power of its centrifugal field. This extraordinary result suggests that a hybrid rotor could fractionate submicrometer solutes to high purity at high process capacity. However, a number of obstacles, in particular the stability of the carrier flow and the solute path lines, may hinder development of this rotor. This paper describes the principles underlying the design and operation of a hybrid rotor, as well as some of the problems that will have to be resolved before this technology can be reduced to practice.
Processing of Fusion Proteins with Immobilized Factor Xa
Authors: Assouline Zahra , Graham Roger , Miller Robert C. , Warren R. Antony J. , Kilburn Douglas G. 
Published: March 2005

AbstractFactor Xa, with a cellulose‐binding domain (CBD) fused to the C‐terminus of the heavy chain (FXa‐CBD), is active in solution and when immobilized on cellulose. A second derivative of factor Xa in which a hexahistidine tail is fused to the C‐terminus of the heavy chain (FXa‐H6) also retains activity when immobilized, in this case on Ni2+‐NTA agarose. The stabilities and activities of FXa‐CBD and FXa‐H6 immobilized on cellulose and Ni2+‐NTA agarose, respectively, are similar. Immobilized factor Xa derivatives can be used to remove affinity tags from appropriate fusion proteins without contaminating the desired product with factor Xa.
Computer Simulation of Low‐Density Lipoprotein Removal in the Presence of a Bioreactor Containing Phospholipase A2
Authors: Shefer Samuel D. , Breslau Joshua , Langer Robert 
Published: March 2005

AbstractHigh concentrations of low‐density lipoproteins (LDL) in the blood can lead to coronary heart disease, the primary cause of death in the Western hemisphere. A new treatment to reduce LDL levels is now being tested on rabbits, which are model animals for hypercholesteremia. The treatment involves using an immobilized enzyme within a bioreactor that is incorporated in an extracorporeal circuit. The enzyme modifies LDL to a form that is much more rapidly removed from the circulation. A mathematical model to describe LDL metabolism in the presence of the bioreactor was developed to give a better understanding of the biodistribution of modified LDL during and following treatment. A four‐compartment model was developed on the basis of previous studies on human lipid metabolism, with the specific values of the constants taken from the experimental data on rabbits. A Macintosh II computer with a Stella II modeling program was used to simulate the treatment and to predict LDL levels over time given different values for initial enzyme activity, length of treatment, rate of enzyme denaturation, and other relevant parameters. The model provided a close fit with the experimental results for the change in total cholesterol. It confirmed the observed delay in the plasma cholesterol rebound level after the end of the extracorporeal treatment. One conclusion derived from both the experimental data and the model is that during the first 1.5 h, the limiting step for LDL removal is the rate at which modified LDL is taken up by the liver. However, bioreactor cessation becomes the limiting step in maintaining low LDL levels for an extended term. The study suggests that continuous modification of LDL, possibly using an implantable device, is required to maintain low levels of plasma LDL.
A Simple Model To Predict the Effectiveness of Molecules That Block Attachment of Human Rhinoviruses and Other Viruses
Authors: Wickham Thomas J. , Shuler Michael L. , Hammer Daniel A. 
Published: March 2005

AbstractThe binding of viruses to cell surfaces is often mediated by cell surface receptors. The use of soluble receptors, such as intracellular adhesion molecule‐1 (ICAM‐1) for human rhinovirus (HRV), CD4 for human immunodeficiency virus (HIV), and CR2 for Epstein‐Barr virus, for in vivo antiviral therapy is under serious investigation. A number of synthetic compounds that affect HRV attachment and uncoating (termed WIN compounds) are also being studied. However, the mechanism behind the dose‐response effect of these agents in preventing infection has not been clearly demonstrated. In addition to simple blocking (by receptors) or inactivation of binding sites (by WIN compounds) on the virus surface, other mechanisms of inhibition have been proposed and demonstrated, including cooperative inactivation of neighboring sites, receptor‐induced viral attachment protein (VAP) shedding, virus particle inactivation, and inhibition of multivalent virus binding. We present a simple mathematical model to predict the effect of these molecules on virus infection by incorporating only the blocking or site inactivation step of the blocking molecule and its resulting effect on attachment. The ability of the model to reproduce the response of a virus to a dose of blocking molecules is used to distinguish the role of blockers in inhibiting attachment from the other mechanisms of viral inactivation that have been proposed. The model includes both the reversible attachment of the virus to its cellular receptor and to soluble receptors or synthetic molecules (blockers). The model qualitatively predicts the published dose‐response curves for the HRV‐14/WIN, HRV‐14/ICAM‐1, and HIV/ CD4 systems under pseudoequilibrium conditions and is quantitatively accurate as a model for HRV‐14 attachment, consistent with a mechanism involving simple competition of the blocker with the cellular receptor for attachment sites on the virus surface. The failure of the model to properly quantify blocking for EBV and HIV under certain conditions suggests that other effects not included in the model are important for viral entry in these systems.
Dichloroethane Removal from Gas Streams by an Extractive Membrane Bioreactor
Authors: Freitas Santos Luisa M. Dos , Hömmerich Uwe , Livingston Andrew G. 
Published: March 2005

AbstractA novel extractive membrane bioreactor (EMB) system, in which a silicone rubber membrane separates a contaminated gas stream from an aerated biomedium, extracted and biodegraded 1,2‐dichloroethane (DCE) from a gaseous waste stream. Selective transfer of the organic compound takes place through the silicone membrane to the biomedium, where biodegradation occurs. At a gas flow rate of 770 mL min−1 and an average DCE concentration of 0.65 mg L−1, the EMB, with 2.5 m2 of membrane surface contained in a spirally wound module, degraded 91% of the DCE. A biofilm grew on the membrane surface and caused a 600 mbar pressure drop at steady state conditions. The chloride ions released by DCE mineralization were 99% of the stoichiometric amount, while 60% of the total amount of carbon crossing the membrane as DCE was bioconverted to CO2. The overall mass transfer coefficient across the membrane in the absence of microbial activity was (0.8‐1.3) × 10−6 m s−1 and was controlled by the liquid film resistance. Extensive biofilm growth significantly lowered the DCE flux. Comparison of the EMB to a direct bioscrubbing process showed that the EMB system has superior volumetric efficiency.
Membrane‐Based Cell Affinity Chromatography To Retrieve Viable Cells
Authors: Mandrusov Evgenia , Houng Abraham , Klein Elias , Leonard Edward F. 
Published: March 2005

AbstractA novel scheme for the separation and live recovery of one cell type from a mixture of cells using a cell affinity chromatography (CAC) system is demonstrated. An antimurine IgG was chemically immobilized to a cellophane support via a carbonyldiimidazole (CDI) link. Murine splenocytes flowed over the support, and B‐cells were allowed to attach at a shear rate of 15 s−1. Once loading was terminated, the support was washed at a shear rate of 315 s−1 to remove nonspecifically bound cells. Elution of the B‐cells was initiated by the transmembrane diffusion of hydrochloric acid (pH 1), supplied to the side of the membrane opposite the cells. At the same time, a shear flow of normal saline was established on the cell side of the membrane, and cells, freed by acid, were retrieved. Results showed that, on average, 250 cells/mm2 attached to antibody immobilized on cellophane surfaces, at a shear rate of 15 s−1, and that attached cells were successfully displaced by acid supplied to the side of the membrane opposite that holding the cells. On average, at least 60% of the B‐cells removed by this elution appeared viable, based on a Trypan Blue dye exclusion assay.
Extraction of Charged Fusion Proteins in Reversed Micelles: Comparison between Different Surfactant Systems
Authors: Forney Craig E. , Glatz Charles E. 
Published: March 2005

AbstractBehavior of a series of fusion proteins of varying charge in reversed micellar extraction was studied. The proteins consisted of the enzyme glucoamylase from Aspergillus awamori joined to short peptides containing from 0–10 additional aspartate residues. The fusions were partitioned into two different cationic surfactant systems, one based on the surfactant trioctylmethylammonium chloride (TOMAC) and the other on cetyltrimethylammonium bromide (CTAB). These two systems differed chiefly in micelle size as measured by the water to surfactant ratio, w0. Water numbers were determined for the TOMAC system, with values of approximately 10, and as a function of pH and ionic strength for CTAB for each of the mutant enzymes. For the CTAB system, water numbers were as low as 50 with NaCl concentrations of 500 mM and as high as 68 at 300 mM NaCl (95% confidence level of 2.4). The enzyme partitioned most strongly using CTAB, with maximal recoveries approaching 95%. However, in the CTAB system, there were no significant differences in behavior between the mutants because of the relatively large micellar size, even under high salt concentrations. Extraction of the control enzyme from clarified cell broth indicated that broth components did not significantly interfere with partitioning.
Investigation of the Ethanol Tolerance of Clostridium thermosaccharolyticum in Continuous Culture
Authors: Baskaran Sunitha , Ahn Hyung ‐Jun , Lynd Lee R. 
Published: March 2005

AbstractThe ethanol tolerance of Clostridium thermosaccharolyticum HG8 has been studied in continuous culture using a new technique that requires knowledge of the kinetic constants and measurement of substrate concentrations at various concentrations of the inhibitor. Endogenously produced ethanol was supplemented with exogenously supplied ethanol to achieve various inhibitor concentrations. The ethanol tolerance of C. thermosaccharolyticum was significantly greater than expected on the basis of most previous reports, which may be explained in part by acclimation occurring over time periods exceeding those typical of batch systems studied previously. An ethanol concentration of 40 g/L is required for 50% growth inhibition of C. thermosaccharolyticum at 55 °C. Process considerations suggest that the ethanol tolerance of C. thermosaccharolyticum is unlikely to significantly constrain its use for ethanol production from cellulosic biomass. Ester‐linked phospholipid membrane analyses (ELPFA) revealed that growth in the presence of high concentrations of ethanol (33 g/L) resulted in a membrane profile having increased fluidity and molecular diversity. Ethanol‐induced changes included a significant increase in shorter chain unsaturated fatty acids (C15:0) at the expense of longer chain unsaturated fatty acids (C17:0) and a slight increase in the amount of mono‐unsaturated fatty acids.
Metabolic Control Analysis of the Penicillin Biosynthetic Pathway in a High‐Yielding Strain of Penicillium chrysogenum
Authors: Nielsen Jens , Jørgensen Henrik S. 
Published: March 2005

AbstractMetabolic control analysis is used to identify the rate‐limiting step in the penicillin biosynthetic pathway in Penicillium chrysogenum. The analysis is carried out using a kinetic model for the first two steps in the pathway, i. e., the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS). The kinetic model is based on Michaelis—Menten type kinetics, with noncompetitive inhibition of the ACVS by ACV and competitive inhibition of the IPNS by glutathione. From measurements of the enzyme activities and intracellular metabolites during a fed‐batch cultivation, the kinetic model is used to predict the flux through the pathway. The model prediction corresponds well with the measured rate of penicillin biosynthesis. From measurement of the activity of the acyl‐CoA:isopenicillin acyltransferase, which catalyzes the third and last reaction in the pathway, it is concluded that the rate‐limiting step is either at the ACVS or at the IPNS. From the kinetic model, the elasticity coefficients and the flux control coefficients are calculated throughout the fed‐batch cultivations, and it is found that there is a shift in the flux control from the ACVS to. the IPNS during the cultivation.
Tracking of Individual Cell Cohorts in Asynchronous Saccharomyces cerevisiae Populations
Authors: Porro Danilo , Srienc Friedrich 
Published: March 2005

AbstractA novel flow cytometric procedure has been developed with the aim to obtain the growth properties of individual Saccharomyces cerevisiae cells in asynchronous culture. The method is based on labeling of the cell surface with FITC‐conjugated concanavalin A and detection of the single‐cell fluorescence with flow cytometry after cell exposure to growth conditions. Because the formation of new cell wall material in budded cells is restricted to the bud tip, exposure of the stained cells to growth conditions results in three cell types: (i) stained cells, (ii) partially stained cells, and (iii) unstained cells Analysis of the staining pattern over time permits the determination of the specifice growth rate of the cell population, the length of the budded cell cycle phase, and the growth pattern during the cell cycle of newly formed, partially stained daughter cells. The procedure has been tested with yeast cell populations growing at different rates. The data suggest an exponential increase in the size of individual cells during the cell cycle, as reflected by the forward angle light scattering (FALS) signals. It has been found that the apparent single‐cell specific cell size growth rates, determined by FALS intensity, are significantly lower than the specific growth rates of the overall population. This could indicate that the tracking of a cohort of cells is significantly perturbed by a distribution of staining levels of daughter cells at cell division and that FALS may not be a good indicator of the cell size.
Process Characterization of a Novel Cross‐Regulation System for Cloned Protein Production in Escherichia coli
Authors: Chen Wilfred , Kallio Pauli T. , Bailey James E. 
Published: March 2005

AbstractA novel cross‐regulation expression system has been shown previously to be very effective for regulated recombinant protein production. Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors. Using production of cloned chloramphenicol acetyltransferase (CAT) as a model system, several factors determining the performance of this system were examined. Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT production were examined. The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase. Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level. CAT message levels were minimal preinduction and far above background postinduction, consistent with prior simulation results. Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10‐fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate. Maintenance of significant CAT message levels with a concomitant 2‐fold increase in CAT accumulation was achieved by extending cell erowth in a fed‐batch process.