bluuebunny/crossref_metadata_2025 huggingface dataset:
St Aldhelm's bees (De uirginitate prosa cc. IV-VI): some observations on a literary tradition
Authors: Casiday Augustine
Published: March 2005

Although the classic comparison of monks to bees owes its enduring success chiefly to the Vita S. Antonii, one of the most interesting developments of that simile is found in the prose treatise De uirginitate by Aldhelm of Malmesbury. In his writings, Aldhelm demonstrates familiarity with most of the conventional similes – monks are like bees in their industry, their intelligence, their chastity, and so on – but he also insists that monks are like bees in their ‘voluntary solidarity’ and obedience to leadership. This is a novel claim, one that I will argue Aldhelm makes by introducing a theme known from other Christian (and pagan) literature into his advice to nuns. The present article will describe the traditions incorporated by Aldhelm into his claim that monks, like bees, are obedient to a fault. In this way, this article will offer a broad view of the literary heritage to which Aldhelm's treatise belongs and in which it should be interpreted. This will entail an assessment of which sources Aldhelm likely knew. While this assessment is indebted to the excellent notes by Rudolf Ehwald (as indeed all scholarship subsequent to Ehwald must be),it will not be bound by Ehwald's conclusions. In some instances, I will posit sources not mentioned by, and perhaps not detected by, Ehwald; in others, I will with trepidation suggest refinements to Ehwald's work. It is hoped that on these grounds the article will be useful to students both of late antique monasticism and of Anglo-Saxon England. Since this is the goal of the article, it will be convenient to begin each section with an excerpt from Aldhelm and follow it with the relevant antecedents; each section will then be concluded with a return to Aldhelm; this will allow us to appreciate the distinctiveness of Aldhelm's contribution. The article itself will be concluded with an overview of the comparisons and of the relationship between the earlier writings and Aldhelm's.
Partners Apart? The Foreign Policy Attitudes of the American and European Publics
Authors: ISERNIA PIERANGELO , EVERTS PHILIP P.
Published: March 2005

It is often argued today that a deep and troublesome gap across the Atlantic has been developing and that Europeans and Americans no longer share the same view of the world. On the basis of data gathered in the 2002 Transatlantic Trends Survey, held in the USA and six European countries, this article assesses whether there is indeed such a gap at the mass level. It focuses on three major dimensions of world views: (1) perceptions of threats (2) the sense of affinity with other countries in terms of allies, friends or foes, and (3) attitudes toward the use of force, both in general and in specific circumstances, more particularly the war over Iraq. It concludes that European publics in 2002 looked at the world in a way that is rather similar to that of many ordinary Americans including harbouring deep reservations about the conduct of certain aspects of U.S. foreign policy. Both publics share fundamental worldviews. On Iraq, Europeans and Americans agreed in some respects (such as the necessary role of the UN) but disagreed on other. In many respects at the mass level the differences across the Atlantic are of degree, and not fundamental. They result from disaffection with the present administration rather than with US policies in general. Moreover, the alleged European ‘anti-Americanism’ is a misnomer, which hides the considerable sympathies and warm feelings towards America, and the perceived common interests and values.
Continuous Recombinant Bacterial Fermentations Utilizing Selective Flocculation and Recycle
Authors: Henry Kimberly L. , Davis Robert H. , Taylor Austin L.
Published: March 2005

Selective recycle has successfully been used to maintain an unstable plasmid‐bearing bacterial strain as dominant in a continuous reactor, whereas the culture reverts to 100% segregant cells when selective recycle is not used. The plasmid‐bearing strain is slower growing and flocculent; however, when the cells lose their plasmid, the resulting segregant cells are nonflocculent and grow at a faster rate due to their decreased metabolic burden. Both types of cells exit a chemostat and enter an inclined settler where the flocculent plasmid‐bearing cells are separated from the nonflocculent segregant cells by differential sedimentation. The underflow from the cell separator, which is enriched with plasmid‐bearing cells, is recycled back to the chemostat, while the segregant cells are withdrawn off the top of the setter and discarded. The experimental results agree well with selective recycle reactor theory. On the basis of the theory, a criterion is presented that has been shown to successfully predict whether or not a selective recycle reactor can maintain a plasmid‐bearing strain.
Experimental Collision Efficiencies of Polymer‐Flocculated Animal Cells
Authors: Auninst John G. , Wang D. I. C.
Published: March 2005

The determination of particle collision kinetics is useful to decouple the effects of process parameters on individual events in flocculation. This paper discusses the effects of flocculation conditions on the collision efficiency of ATCC strain CRL 1606 hybridomas flocculated with poly‐L‐histidine. Experimental determinations of the collision efficiency of cells in Couette flow are presented over a range of experimental conditions. The collision efficiency correlates with the cell ζ potential to the ‐2.4 power at high surface coverage, consistent with literature results in latex systems. At low coverage, accounting for the distribution of polymer on the cells corrects for deviation from the high‐coverage behavior. Collision is dependent on the hydrodynamic environment as well. At high surface coverage, collision efficiency is weakly dependent on hydrodynamic conditions and follows a dependency on the shear rate and viscosity to the ‐0.32 power. This is consistent with ionic coagulation theory. At low surface coverage, the collision efficiency is strongly dependent on the viscous fluid forces. The results versus both dose and shear rate over the entire range of surface coverages are consistent with weak intercell bonding. Collision kinetics in the presence of high molecular weight dextrans show steric hindrance to cell collision.
Genetic Engineering Approach to Toxic Waste Management: Case Study for Organophosphate Waste Treatment
Authors: Coppella Steven J. , Delacruz Neslihan , Payne Gregory F. , Pogell Burton M. , Speedie Marilyn K. , Karns Jeffrey S. , Sybert Edward M. , Connor Michael A.
Published: March 2005

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled‐up to the pilot scale. The cell‐free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.
Effect of Preinduction Specific Growth Rate on Recombinant Alpha Consensus Interferon Synthesis in Escherichia coli
Authors: Curless C. , Pope J. , Tsai L.
Published: March 2005

The effect of preinduction specific growth rate on the yield of recombinant alpha consensus interferon in Escherichia coli K‐12 was investigated. The cells used in the investigation contain a temperature‐sensitive, walkaway plasmid bearing an insert that codes for alpha consensus interferon. Transcription of the recombinant gene is controlled by a lambda repressor/pL promoter system. The lambda promoter is regulated by the temperature‐sensitive gene cl857 at 30 °C, but at 42 °C the promoter is derepressed. The cells were grown under glucose‐limited conditions in a chemostat at pH 7 and 30 °C. Once steady state was achieved, the vessel temperature was raised to 42 °C and a fed‐batch mode was initiated. Six dilution rates ranging from 0.025/h to 0.2/h were investigated. Cell dry weight, alpha consensus interferon content, glucose concentration, acetate concentration, and plasmid stability were measured. At each dilution rate, the expression level of alpha consensus interferon (g/g of cell dry wt) reached its maximum value approximately 3 h after induction. In addition, the expression level of alpha consensus interferon increases 4‐fold as the dilution rate prior to induction is increased from 0.025/h to 0.2/h. Consequently, the expression of recombinant protein produced by E. coli is dependent on the preinduction specific growth rate.
Production of Monoclonal Antibodies by Hybridoma Cells in a Flat Sheet Membrane Bioreactor
Authors: Hagedorn J. , Kargi F.
Published: March 2005

The purpose of this study was to investigate hybridoma growth and monoclonal antibody formation in a flat sheet membrane bioreactor. The effects of several different molecular weight cutoff membranes on growth and antibody formation were investigated. Nutrient and toxic product diffusion through the membranes were quantified, and the kinetic and physical constants of the system were determined.
Biological Production of Acrylic Acid from Cheese Whey by Resting Cells of Clostridium propionicum
Authors: O'Brien Dennis J. , Panzer Curtis C. , Eisele William P.
Published: March 2005

Biological production of acrylic acid from cheese whey has been demonstrated. Lactose in sweet whey fortified with yeast extract was first converted to a stoichiometric mixture of propionic and acetic acids in 70 h by a coculture of Lactobacillus bulgaricus and Pro‐pionibacterium shermanii. Further conversion of propionate to acrylate was accomplished by resting cells of Clostridium propionicum in systems in which methylene blue acted as an electron acceptor. A maximum acrylate yield of 0.133 mmol/g of wet cell was achieved for periods of 6 h, after which resting cell activity declined substantially. Bio‐conversion of propionate to acrylate could also be carried out by cells immobilized in calcium alginate beads with no decrease in cell productivity or initial reaction rate.
Precipitation of Nucleic Acids with Poly (ethyleneimine)
Authors: Cordes Richard M. , Sims W. Bradley , Glatz Charles E.
Published: March 2005

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly (ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95 % removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.
Separation of β‐Carotene Mixtures Precipitated from Liquid Solvents with High‐Pressure CO2
Authors: Chang Chiehming J. , Randolph Alan D. , Craft Neal E.
Published: March 2005

Solids precipitation from liquid solvents, with dissolution by high‐pressure CO2 as an antisolvent to create supersaturation, is a potentially attractive crystallization process. Solids can be recrystallized and easily isolated from the liquid solvent. The gas antisolvent solvent (GAS) process was used to separate and purify β‐carotene from a mixture containing carotene oxidation products. Total β‐carotene was successfully separated from oxides, and an enriched traras‐β‐carotene was obtained from its cis isomers. The separation was carried out in both batch and continuous modes. Relative solubility of the analytes and the antisolvent (CO2) have a dramatic influence on the absolute yield and purity of the product.
Synergistic Response of Plant Hairy‐Root Cultures to Phosphate Limitation and Fungal Elicitation
Authors: Dunlop David S , Curtis Wayne R
Published: March 2005

The combined effects of phosphate limitation and fungal elicitation on sesquiterpene production were examined in Agrofeacterium‐transformed “hairy‐root” cultures of Hyo‐scyamus muticus Limiting the initial supply of inorganic phosphate to elicited cultures resulted in a 4.2‐fold increase in solavetivone production as compared to elicitation at control media phosphate levels (1.1 mM). Because growth was attenuated at low initial phosphate levels, production per unit cell mass increased 46 times as compared to the control. Both elicited and nonelicited cultures showed enhanced solavetivone production at lower initial phosphate levels. In fact, the specific productivity of nonelicited roots grown in phosphate‐free media was comparable to the specific productivity of fungally elicited roots that are not phosphate limited. Addition of fungal elicitors enhanced specific productivity of solavetivone about 200‐fold over nonelicited cultures at nearly all phosphate levels. Phosphate limitation and fungal elicitation are therefore synergistic, and optimal total production is achieved by simultaneous application of these two production strategies.
Filtration Properties of Mycelial Microbial Broths
Authors: Oolman Timothy , Liu Tuan‐Chi
Published: March 2005

The filtration properties of three mycelial cultures (Streptomyces griseus, Streptomyces tendae, and Penicillium chrysogenum) have been measured and quantified in terms of standard filtration equations. Three of the filtration parameters, the hyphal density, the index of compressibility, and the Kozeny constant, were found to vary systematically with broth age and with the visually observed morphology (i.e., pellets vs filaments). Since broth age and mycelial morphology both have a strong influence on bioreactor productivity, it is suggested that filtration could be used for on‐line monitoring of mycelial fermentations.
Effects of Oxygen and Carbon Dioxide Partial Pressures on Monascus Growth and Pigment Production in Solid‐State Fermentations
Authors: Han Ohantaek , Mudgett Richard E.
Published: March 2005

Levels of oxygen and carbon dioxide in the gas environment were found to influence pigment production significantly and growth to a lesser extent in solid‐state fermentations with Monascus purpureus on rice. Maximum pigment yields were observed at 0.5 atm of oxygen partial pressure in closed pressure vessels. However, high carbon dioxide partial pressures progressively inhibited pigment production, with complete inhibition at 1.0 atm. In a closed aeration system with a packed‐bed fermentor, oxygen partial pressures ranging from 0.05 to 0.5 atm at constant carbon dioxide partial pressures of 0.02 atm gave high pigment yields with a maximum at 0.50 atm of oxygen, whereas lower carbon dioxide partial pressures at constant oxygen partial pressures of 0.21 atm gave higher pigment yields. Maximum oxygen uptake and carbon dioxide evolution rates were observed at 70–90 and 60–80 h, respectively, depending on the gas environment. Respiratory quotients were close to 1.0, except at 0.05 atm of oxygen and 0.02 atm of carbon dioxide partial pressures. Optimum conditions for pigment formation were generally not the same as those for growth.
Ellipsometric Evaluation of β‐Lactoglobulin Adsorption onto Low‐and High‐Energy Materials
Authors: Al‐Malah Kamal , Mcguire Joseph , Krisdhasima Viwat , Suttiprasit Prasert , Sproull Robert
Published: March 2005

Ellipsometry was used to study solid‐surface energetic and temperature effects on the apparent equilibrium adsorptive behavior exhibited by β‐lactoglobulin. Adsorption isotherms were constructed for β‐lactoglobulin on seven different silicon surfaces chemically modified to exhibit varying hydrophobicities: plateau values of adsorbed mass were observed to increase with increasing degree of solid‐surface silanization with dichlorodimethylsilane. β‐Lactoglobulin isotherms at 2,27, and 52°C were constructed on unsilanized and completely silanized silicon surfaces: adsorbed mass increased upon increasing temperature beyond 2°C, but no difference was observed between isotherms drawn at 27 and 52°C for either surface. Isotherms were then constructed for acrylic, polycarbonate, polyester, glass, and no. 304 stainless steel surfaces at 37 and 55°C. In all cases, optical properties of the protein films were ellipsometrically measured and their adsorbed mass was calculated from film thickness and refractive index. The protein adsorption results recorded for the polymer, glass, and stainless steel surfaces could not be explained with reference to solid‐surface hydrophobicity alone. For polymers, adsorbed mass is suggested to be related to the extensibility of molecular structure at the surface. Glass was observed to adsorb the greatest mass of β‐lactoglobulin, and no. 304 stainless steel the lowest; however, glass and the polymers, being transparent, presented a different optical problem for ellipsometry compared to that posed by silicon and stainless steel. A temperature dependence of β‐lactoglobulin adsorption was not observed between 37 and 55°C, probably due in part to the temperatures studied having been below the denaturation temperature of β‐lactoglobulin, as indicated by differential scanning calorimetry.
Posttranslational Limitations to Methanol Oxidase Activity in the Methylotrophic Yeast Hansenula polymorphs As Determined from Single Growth Rate Perturbations
Authors: Bryers J. D. , Westcott R. W. , Sayles G. D.
Published: March 2005

Single shifts in growth rate were imposed upon continuous cultures of the methylotrophic yeast Hansensula polymorpha growing under carbon limitation on 4 g/L methanol and 1 g/L glucose. Concentrations of system level parameters (glucose, methanol, and cell biomass) and intracellular components (methanol oxidase and hex‐okinase specific activity, methanol oxidase specific mRNA content, and the ratio of flavin adenine dinucleotide bound to methanol oxidase) suggest that the reduction in methanol oxidase specific activity upon a shift to a higher growth rate is limited, not at the transcription level, but rather posttranslationally during peroxisome assembly. Subsequent [35S] methionine pulse‐chase techniques and immunoprecipitation of methanol oxidase monomers and octamers indicate, upon a dilution rate shift from 0.2 to 0.35 h−1, that monomeric methanol oxidase fails to polymerize, terminating peroxisome formation. Those peroxisomes formed at the lower growth rate slowly disintegrate at the higher growth rate, releasing bound flavin adenine dinucleotide.
Acoustic Resonances in Chicken Eggs
Authors: Sinha Dipen N. , Johnston Roger G. , Grace W. Kevin , Lemanski Cheryl L.
Published: March 2005

We report the first acoustical vibration studies on chicken eggs. Measurements of the acoustic resonances offer the potential of simple, noninvasive, and rapid characterization of eggs. The technique appears at least partially sensitive to yolk puncture, shell cracks, and the presence of Salmonella bacteria. There may be practical quality control applications.
Use of Monoclonal Anti‐enzyme Antibodies for Analytical Purposes
Authors: Gunaratna P. Chandrani , Wilson George S.
Published: March 2005

This review discusses the analytical applications of monoclonal antibodies specific for enzymes. One important, but not well‐studied, application of these monoclonal antibodies is their use in immobilizing enzymes on solid supports. This method is based on binding the enzymes to an immobilized antibody through the antigen bindng site of the antibody. Enzymes immobilized this way retain much of their activity. The utility of immobilized enzyme reactors prepared by immobilizing the enzymes through antibodies is demonstrated by using them in the determination of acetylcholine and choline in brain tissue extracts. Currently available methods for immobilizing antibodies and enzymes are reviewed. Other issues discussed in this review include the problems and advantages of immobilized enzyme reactors, especially when used in conjunction with HPLC. In addition, the applications of monoclonal antibodies for the detection and measurement of enzymes and their isoforms are summarized.
Spatial Distribution of Mammalian Cells Grown on Macroporous Microcarriers with Improved Attachment Kinetics
Authors: Lim Hyun‐Soo , Han Bong‐Kwan , Kim Jung‐Hoe , Peshwa Madhusudan V. , Hu Wei‐Shou
Published: March 2005

Vero and HepG2 cells were cultivated on macroporous gelatin microcarriers prepared by the calcium carbonate inclusion method. Cell attachment to these microcarriers was slow. For HepG2 cells the subsequent growth was poor. Modification of the microcarriers by incorporation of (diethylamino)ethyl‐HCl improved HepG2 attachment and subsequent growth. Optical sectioning with confocal microscopy allowed visualization of the distribution of cells within microcarriers. In most microcarriers, cells were found to preferentially populate regions close to the external surface and some cavities in the interior. Despite the incomplete occupancy of the interior of the microcarriers, high cell concentrations were achieved.
Isolation of Vindoline from Catharanthus roseus by Supercritical Fluid Extraction
Authors: Song Kyu‐Min , Park Sang‐Woo , Hong Won‐Hi , Lee Huen , Kwak Sang‐Soo , Liu Jang‐Ryol
Published: March 2005

Vindoline was extracted from the leaves of Catharanthus roseus over the ranges of 35–70 °C and 100–300 bar using supercritical carbon dioxide with and without the addition of 3 wt % ethanol as a cosolvent. The vindoline contents in the extracts were determined by HPLC and identified by LC/MS. The remarkable highest vindoline concentration, 58 wt %, was obtained at the lowest temperature, 35 °C, and the highest pressure, 300 bar, of this study. The use of a cosolvent only slightly improved the extraction yields or selectivities at some experimental conditions.
TOPICAL PAPER: Utilization of Hairy Root Cultures for Production of Secondary Metabolites
Authors: Toivonen Leena
Published: March 2005

The possibility of producing useful chemicals by plant cell cultures has been studied intensively for the past 30 years. However, problems associated with low product yields and culture instabilities have restricted wider industrial application of plant cell culture. The employment of hairy root culture technology, developed in the past 10 years, offers new opportunities for in vitro production of plant secondary metabolites. In contrast to cell suspension cultures, hairy root cultures are characterized by high biosynthetic capacity and genetic as well as biochemical stability. In this review, the establishment and cultivation of hairy root cultures as well as their properties and application for production of secondary metabolites are discussed.
Performance of a Packed Column for Continuous Supercritical Carbon Dioxide Processing of Anhydrous Milk Fat
Authors: Bhaskar Ajay R. , Rizvi Syed S. H. , Harriott Peter
Published: March 2005

A continuous system was designed for the processing of anhydrous milk fat (AMF) with supercritical carbon dioxide (SC‐CO2). The packed column was operated as a stripping column with SC‐CO2 as the continuous phase and AMF as the dispersed phase. The extraction was studied at 24.1 MPa and 40 °C. To utilize the functional similarities and make the analysis simpler, the AMF triglycerides were grouped as low‐melting (LMT: C24–C34), medium‐melting (MMT: C36–C40), and high‐melting triglycerides (HMT: C42–C54). There was an increase in the concentrations of LMT and MMT and a decrease in that of HMT in the extract. The raffinate has a high concentration of HMT with trace amounts of LMT. The performance characteristics of the packed column were described by the number of transfer units (Nog), the overall volumetric mass transfer coefficient (KOGa), and the height of a transfer unit (HOG). The NOG and Kogci values decreased and the HOG value increased with increasing solvent‐to‐feed ratio. The HOG values increased and the NOG and KOGa values decreased from LMT to MMT to HMT, but the HOG values for HMT were greater than expected. The values of NOG, HOG, and KOGa for AMF (as a single component) were intermediate to those of MMT and HMT.
Characteristics of Batch Suspension Cultures of Preconditioned Coleus blumei Cells: Sucrose Effect
Authors: Martinez Blanca C. , Park Chang‐Ho
Published: March 2005

Batch suspension cultures of Coleus blumei cells preconditioned with 0.1% dimethyl sulfoxide (DMSO) maintained viability at above 85 % during a 14‐day culturing period for initial sucrose concentrations of 30–70 g/L. At 60 g/L sucrose, cells grew rapidly with a doubling time of 10.7 h, and dry cell mass reached a maximum of 15.6 g/L. Broad optima of cell yield (0.46–0.48 g of cell/g of sucrose), rosmarinic acid (RA) yield (3.1–3.5 g of RA/100 g of sucrose), and rosmarinic acid production (1.0–1.1 g of RA/L) were observed at 40–60 g/L sucrose concentration. Sucrose in the medium was hydrolyzed to glucose and fructose by extracellular and/or cell‐bound invertase, and glucose was taken up by the cells at a higher rate than fructose. Even at sucrose concentrations of 40–70 g/L, sucrose consumption over 14 days was limited to 33 g/L.
Extraction of Cytochrome c in Sodium Dodecylbenzenesulfonate Microemulsions
Authors: Jolivalt Claude , Minier Michel , Renon Henri
Published: March 2005

The partition of cytochrome c between an aqueous phase in equilibrium with a microemulsion composed of sodium dodecylbenzenesulfonate, butanol, and decane was studied. By adjusting the pH and the ionic strength in the aqueous phase, cytochrome c can be quantitatively extracted in the microemulsion as long as the protein concentration in the organic phase is less than the initial concentration of micelles. Above this limit, an intermediate phase, containing part surfactant and part cytochrome c, is located between the aqueous and microemulsion phases. The role of electrostatic interactions in the protein partitioning is shown by varying the composition of the phases (pH, salinity, surfactant, and cosurfactant concentrations) and then discussed. Extraction depends on the relative values of the pH of the aqueous phase and the pI of cytochrome c, the size of the micelles, and the protonation of acidic residues. Cytochrome c can be recovered in an aqueous phase by adding butanol in the microemulsion phase. In such conditions, cytochrome c retains 90% of its initial activity, as measured before the extraction step.
Utilization of Microwaves in the Study of Reaction Kinetics in Liquid and Semisolid Media
Authors: Welt B. A. , Steet J. A. , Tong C. H. , Rossen J. L. , Lund D. B.
Published: March 2005

An apparatus, referred to as the microwave kinetics reactor (MWKR), incorporating a microwave‐transparent pressure vessel with mechanical mixing together with a microwave temperature controller, was developed and tested. Experiments were performed to determine a safe and practical operating envelope for the MWKR. Temperatures measured at the center and at the top surface of varying viscosity solutions in the MWKR were used to determine the adequacy of mixing when the solutions were heated from room temperature to 120 °C and held for a prescribed period of time. The viscosities of the solutions were adjusted by adding (carboxymethyl)cellulose (CMC) to distilled water. A 2% CMC solution represented the upper viscosity limit to be heated homogeneously in the apparatus (approximately 2000 cP). To illustrate the usefulness of this apparatus in obtaining kinetic parameters, thiamin hydrochloride degradation kinetics in pea puree were determined at 100, 120, and 130 °C and compared to those obtained from the literature. The MWKR offers an attractive, efficient, convenient, and accurate alternative for the study of reaction kinetics in viscous fluids and semisolids because of its short come‐up time and mixing capability. Experimentally determined kinetic parameters compared very well to those obtained from the literature. As expected the kinetics was first‐order, with an activation energy, Ea, of 27.0 kcal/mol.
Molecular Characterization of β‐Lactamase Inclusion Bodies Produced in Escherichia coli. 1. Composition
Authors: Valax Pascal , Georgiou George
Published: March 2005

We have determined the macromolecular composition of inclusion bodies formed by overexpressing β‐lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition. Specifically, contaminating polypeptides ranged from less than 5% to over 50% of the total protein content. Phospholipids composed 0.5–13 % of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature β‐lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown at basic pH. Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.
Effects of the par Locus on the Growth Rate and Structural Stability of Recombinant Cells
Authors: Kim Jeong‐Yoon , Kang Hyun Ah , Ryu Dewey D. Y.
Published: March 2005

A decreased growth rate of recombinant cells is observed when a cloned gene protein encoded in a multicopy plasmid is induced from a strong promoter. This negative effect on the cell growth rate may be the consequence of alternate use or reallocation of energy, precursor metabolites, and protein synthesizing machinery. In order to analyze the causes for this adverse effect, we have studied how genetic elements present in multicopy plasmids affect the growth ofrecombinant Escherichia coli (K12ΔH1 ΔtrpEA). Turning on of the PL promoter, whether or not protein‐coding sequences were present downstream of the promoter, decreased the growth of the recombinant cells by 15–50 %. This result suggests that the essential factor(s) for cell growth may be sequestered by the genetic elements present in the plasmids and thus may cause the decreased growth. The negative effect of the de‐repressed PL promoters on cell growth was partially reversed when the par sequence from pSC101 was inserted into the same plasmid. In each case when the plasmid carried only the PL promoter, the PL promoter followed by the N‐terminal part of the protein‐coding region, or the PL promoter followed by complete sequences encoding a protein, the par sequence exhibited a positive effect on the growth rate of the recombinant cells, which usually grow at a slower rate than the host cell. In addition, it was found that the recombinant cells bearing the plasmid with the par locus were as resistant as the host cell to osmotic shock when exposed to distilled water, whereas the recombinant cells bearing the plasmid without the par locus were sensitive to osmotic shock and resulted in lysis.
Copy Number Modulation in an Autoselection System for Stable Plasmid Maintenance in Saccharomyces cerevisiae
Authors: Compagno C. , Tura A. , Ranzi B. M. , Alberghina L. , Martegani E.
Published: March 2005

Efficient expression of a foreign gene requires a stable vector present at a high number of copies per cell. We have constructed an autoselection system for the stable maintenance of expression vector in the yeast Saccharomyces cerevisiae that uses the fructose 1, 6‐bisphosphate aldolase gene (FBA1) to stabilize plasmids in cells bearing a disruption of the chromosomal FBA1 gene. This system allowed us to obtain stable production of a reporter heterologous enzyme (Escherichia coli β‐galactosidase) in rich media. By using an inducible promoter to regulate the expression of FBA1 gene, we have also obtained the modulation of plasmid copy number by carbon source.
Growth versus Function in the Three‐Dimensional Culture of Single and Aggregated Hepatocytes within Collagen Gels
Authors: Parsons‐Wingerter Patricia A. , Saltzman W. Mark
Published: March 2005

Hepatocytes require cell—cell and cell—substrate interactions to survive and function. Furthermore, cell proliferation and differentiated function are often reciprocally related activities, both of which are highly regulated in the liver microenvironment. To mimic the physiological organization of hepatocytes into multicellular plates, small hepatocyte aggregates containing an average of 4–6 cells were formed. The aggregates were suspended within three‐dimensional collagen gels and compared to both single hepatocytes (suspended within collagen gels) and hepatocyte monolayers (on tissue culture polystyrene) over 6 and 10 days in culture. The comparison of aggregates with single cells represents a novel in vitro system for the study of hepatocyte cell‐cell interactions. All hepatocyte‐populated gels displayed almost identical patterns of cell behavior: hyperplastic or hypertrophic growth (indicated by DNA or protein concentration) coincided with the decline of phenotypic function (assessed by uric acid and albumin production), while functional recovery often accompanied the cessation of growth. However, aggregate cultures maintained an average of 2‐fold higher concentrations of functional parameters than singlet cultures when cells were not involved in growth activities. Much higher concentrations of all cell parameters were sustained by gel‐dispersed hepatocytes than by hepatocyte monolayers.
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

This 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

To 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

Dense 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

The 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

The 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

All 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

Factor 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

High 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

The 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.