bluuebunny/crossref_metadata_2025 huggingface dataset:
Perceptions and experience of workplace bullying in five different working populations
Authors: Jennifer Dawn , Cowie Helen , Ananiadou Katerina
Published: October 2003

Whilst aggression in the workplace has been systematically studied in recent years, research into workplace bullying per se still remains quite limited. In this article, we report the findings from an investigation into employees' perceptions of social and organizational work conditions and experiences of bullying at work. Six‐hundred‐seventy‐seven employees from five different working populations (managers, teachers, technicians, call centre operators, and engineers) completed the Workplace Relationships Questionnaire (WRQ). This paper presents the results of the analysis, linking the experiences of bullying and perceptions of social and organizational work conditions. The present findings predictably identify victims and non‐bullied participants, and also indicate the existence of a new category of employee affected by the problem of bullying; bullied/non‐victims. Bullied/non‐victims may provide crucial insights into the ways that company practices and policies impact negatively on the whole workforce. Aggr. Behav. 29:489–496, 2003. © 2003 Wiley‐Liss, Inc.
Influence of executive functioning difficulties on verbal aggression in adolescents: Moderating effects of winning and losing and increasing and decreasing levels of provocation
Authors: Santor Darcy A. , Ingram Adrienne , Kusumakar Vivek
Published: October 2003

We examined the extent to which executive functioning difficulties were related to verbal aggression under conditions of gradually increasing or decreasing provocation over both winning and losing trials. Sixty adolescent boys and girls completed a modified Taylor aggression paradigm in which verbally offensive remarks were sent and received. Results showed (a) that during increasing levels of provocation, verbal aggression increased on both winning and losing trials, (b) that verbal aggression decreased during decreasing levels of provocation but only on losing trials, and (c) that difficulties on two measures of executive functioning were related to verbal aggression, but only in the context of increasing verbal provocation. Individuals with executive functioning difficulties responded with more verbal aggression than did individuals with less severe executive functioning difficulties. Results have implications for the manner in which provocation is studied and for understanding the conditions in which executive functioning difficulties contribute to verbal aggression. Aggr. Behav. 29:475–488, 2003. © 2003 Wiley‐Liss, Inc.
Binding Affinities of Host–Guest, Protein–Ligand, and Protein–Transition‐State Complexes
Authors: Houk K. N. , Leach Andrew G. , Kim Susanna P. , Zhang Xiyun
Published: October 2003

The affinities of hosts—ranging from small synthetic cavitands to large proteins—for organic molecules are well documented. The average association constants for the binding of organic molecules by cyclodextrins, synthetic hosts, and albumins in water, as well as of catalytic antibodies or enzymes for substrates are 103.5±2.5 M−1. Binding affinities are elevated to 108±2 M−1 for the complexation of transition states and biological antigens by antibodies or inhibitors by enzymes, and to 1016±4 M−1 for transition states with enzymes. The origins of the distributions of association constants observed for the broad range of host–guest systems are explored in this Review, and typical approaches to compute and analyze host–guest binding in solution are discussed. In many classes of complexes a rough correlation is found between the binding affinity and the surface area that is buried upon complexation. Enzymes transcend this effect and achieve transition‐state binding much greater than is expected from the surface areas.
Electrochemical and Photochemical Control of Host–Guest Complexation at Surfaces
Authors: Cooke Graeme
Published: October 2003

The application of electrochemistry or photochemistry to modulate supramolecular interactions between host–guest systems in solution is a burgeoning field. In particular, the speed and reversibility associated with electrochemically or photochemically actuated supramolecular interactions has allowed the creation and modulation of novel solution‐based devices. In recent years, great advances have been made in transferring these systems from the solution to the solid state, to facilitate the development of molecular‐electronics components that can operate in unison under the influence of an externally applied stimulus. These studies pave the way for the creation of responsive surfaces with advanced materials and nanotechnology applications.
Crystallographic report: Bis[bis(N,N‐dibenzyldithiocarbamato)zinc(II)](4,4′‐bipyridine)
Authors: Yin Xia , Zhang Weiguang , Fan Jun , Wei Feng Xian , Lai Chian Sing , Tiekink Edward R. T.
Published: October 2003

The centrosymmetric structure of {Zn[S2CN(CH2Ph)2]2}2(4,4′‐bipy) features chelating dithiocarbamate ligands so that a trigonal bipyramidal NS4 coordination geometry for zinc results. Copyright © 2003 John Wiley & Sons, Ltd.
New thermoplastic materials reinforced with cellulose based fibers
Authors: Rodríguez Carlos Andrés , Medina Jorge Alberto , Reinecke Helmut
Published: October 2003

The preparation of new cellulose fiber reinforced thermoplastics is described, and the mechanical properties and water absorption of these materials are measured.

In order to improve the compatibility between cellulosic and plastic materials, different amounts of coupling agents, such as organotitanate compounds, maleic anhydride copolymers and their combinations, are studied. As a filler, pine fiber, rose, carnation or coffee husk is used. Furthermore, the influence of the composition of the composites and the particle size of the filler on the material properties of the thermoplastics are investigated. The surface of the fractured samples is analyzed using scanning electron microscopy (SEM). © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3466–3472, 2003
Spinning fibers from poly(ethylene terephthalate) bottle‐grade waste
Authors: Gurudatt K. , De P. , Rakshit A. K. , Bardhan M. K.
Published: October 2003

Poly(ethylene terephthalate) bottle‐grade (BG) waste was converted into spinnable chips and spun on a laboratory‐scale melt‐spinning apparatus into filaments. Virgin fiber‐grade (FG) polyester chips were blended with BG waste during melt spinning so that the influence of blending on the fiber properties could be studied. Subsequently, the scaling‐up of the process was carried out in a polyester recycling plant so that staple fibers could be obtained. In this part of the study, the spinning of blends of BG waste and FG waste was carried out. The BG waste was found to be superior feed stock for melt processing. Fibers with unique properties were obtained from the BG waste. Staple fibers obtained by the blending of FG and BG waste showed properties different from those of fibers spun from BG waste alone. This study also showed that using blends of BG and FG waste could improve the melt processing and staple‐fiber properties. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3536–3545, 2003
Properties of poly(lactic acid) blends with various starches as affected by physical aging
Authors: Wang Hua , Sun Xiuzhi , Seib Paul
Published: October 2003

Poly(lactic acid) (PLA) and starch are both biodegradable and renewable polymers derived from agricultural feedstock. A previous study showed that a small amount (0.5%) of methylenediphenyl diisocyanate (MDI) could enhance the mechanical properties of starch and PLA blends by improving the interfacial interaction. In this study, blends of PLA (1/1, w/w) and starch with or without MDI were evaluated for thermal and mechanical properties as well as morphology, as affected by physical aging when stored up to 12 months at 25 °C and 50% relative humidity. The blends were prepared by thermally blending PLA with wheat starch, corn starch, and/or high amylose corn starch, with or without MDI. All samples exhibited phenomena of physical aging. The samples with MDI aged more slowly, showing a slower reduction rate of excess enthalpy relaxation, than those without MDI. The mechanical properties decreased slowly as aging proceeded. Microstructure showed a reduced interaction between starch and PLA around the interface with aging. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3683–3689, 2003
Effect of ultrasound on extrusion of polypropylene/ethylene–propylene–diene terpolymer blend: Processing and mechanical properties
Authors: Chen Yingzi , Cao Yurong , Li Huilin
Published: October 2003

The effects of ultrasonic irradiation on extrusion processing and mechanical properties of polypropylene (PP)/ethylene–propylene–diene terpolymer (EPDM) blends are examined. Results show that appropriate irradiation intensity can prominently decrease die pressure and apparent viscosity of the melt, increase output, as well as increase toughness of PP/EPDM blends without harming rigidity. In case the blends are extruded with ultrasonic irradiation twice, the impact strength of the blend rises sharply at 50–100 W ultrasonic intensity, and amounts to more than 900 J/m, 1.5 times as high as that of blend without ultrasonic irradiation. Scanning electron microscopy observation shows that with ultrasonic irradiation, morphology of uniform dispersed EPDM phase and good adhesion between EPDM and PP matrix was formed in PP/EPDM blend. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3519–3525, 2003
XPS study of aged polyaniline films
Authors: Jousseaume V. , Morsli M. , Bonnet A.
Published: October 2003

In this article, a study of the aging of conducting Polyaniline–Polystyrene blends using X‐ray photoelectron spectroscopy (XPS) and UV‐visible‐near IR analysis is presented. The physicochemical results are compared to those obtained by electrical measurements. XPS results confirm the existence of an oxidation process also deduced by the electrical conductivity studies. The N1s and S2p core level spectra decomposition allows to show that a deprotonation process and cyclization of tertiary amine occur during aging. The absorption spectrum shows a decrease of delocalized charges and the apparition of localized polarons after a long aging time. All these mechanisms are responsible of the electrical conductivity decrease observed during aging at elevated temperature. The results are presented for films of PANI–CSA–PSt blends, but the conclusions can be extended to pure conducting PANI–CSA films. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3730–3736, 2003
Synthesis and thermal degradation of poly(n‐butylisocyanate) modified by 1,3‐propanesultone
Authors: Dereszewska Alina , Olayo Roberto , Cardoso Judith
Published: October 2003

Poly(n‐butylisocyanates) were synthesized by anionic homopolymerization with the addition of sulfonate groups in the termination stage. Two types of functional polymers were obtained from different quantities of added 1,3‐propanesultone (PrS). When a stoichiometric proportion of sultone is added, the reaction with poly(n‐butylisocyanate) renders a mix of a nonfunctionalized (PBIC) and a telechelic polymer (PBICT). In the presence of an excess of PrS, the proportion of end‐functionalized chains was higher, and a significant change in their thermal properties was observed. Polymers were characterized by FTIR and NMR, and measurements of intrinsic viscosity were made. Thermal degradation of the polymers was studied under nitrogen atmosphere for various heating rates. The kinetic parameters such as the activation energy and the reaction order were determined by using Ozawa's method. Thermal degradation analyses showed that the degradation processes depend strongly on the concentration of functional groups. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3594–3601, 2003
Study of self‐crosslinking acrylate latex containing fluorine
Authors: Chen Yanjun , Zhang Chaocan , Wang Yifeng , Cheng Shiyuan , Chen Peizhi
Published: October 2003

Self‐crosslinking acrylate latices containing fluorine were prepared by the batch method, with N‐hydroxymethyl acrylamide (NMA) as a crosslinking agent. The structure and component of latex polymers were studied by FTIR, ion‐selective electrode analysis, and DSC. The stabilities of latices, such as storage stability, centrifugal stability, and electrolyte‐resistant stability, were examined. The morphology of the latex particles was characterized by TEM and the surface properties were determined by the Wilhelmy method. The effects of NMA on Tg of the latex polymers, the average particle size of the latices, and the surface properties of the latex films were discussed. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3609–3616, 2003
High‐resolution thermogravimetry of polyethersulfone chips in four atmospheres
Authors: Li Xin‐Gui , Shao Hong‐Tao , Bai He , Huang Mei‐Rong , Zhang Wei
Published: October 2003

Thermal degradation and kinetics of polyethersulfone (PES) chips were studied in air, nitrogen, helium, and argon from room temperature to 790°C by high‐resolution thermogravimetry (TG) at a variable heating rate in response to changes in the sample's degradation rate. In the four atmospheres, a two‐step degradation process in air, argon, and helium or a three‐step degradation process in nitrogen of the PES were found in this investigation. In particular, the three‐step degradation process in nitrogen of the PES revealed by the high‐resolution TG was hardly ever observed by a traditional TG. The initial thermal degradation temperature of the PES increases with the testing atmosphere in the following order: air < argon < helium < nitrogen but the activation energy of the first major degradation of PES increases in a different order: argon < nitrogen < helium < air. The degradation temperature, the temperature at the maximum weight‐loss rate, the maximum weight‐loss rate [(dα/dT)m1 and (dα/dT)m2], char yield at 790°C, and activation energy of the first major degradation process obtained by the high‐resolution TG were compared with those by traditional TG. The PES exhibits the largest (dα/dT)m1 and the greatest char yield at 790°C in helium but the largest (dα/dT)m2 and smallest char yield in air. A significant dependency of the thermal decomposition of the polymers on the physicochemical properties (density, thermal conductivity, and oxidative ability) of the testing atmospheres is elaborated for the first time. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3631–3637, 2003
Tensile fracture and failure behavior of technical flax fibers
Authors: Romhány G. , Karger‐Kocsis J. , Czigány T.
Published: October 2003

The apparent tensile strength of technical flax fibers was determined in single‐fiber tests at various clamping lengths (20, 40, and 80 mm) and the outcome was compared with literature data. It was demonstrated that the strength of flax at each clamping length obeyed the two‐parameter Weibull model. The failure mode and sequence were studied in situ (i.e., during loading) by SEM and acoustic emission (AE). The failure sequence (axial splitting of the technical fiber along its elementary constituents, radial cracking of the elementary fibers, multiple fracture of the elementary fibers) concluded reflected the hierarchical build‐up of the flax bast fibers. To the above failure events AE amplitude ranges were assigned. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3638–3645, 2003
Human severe combined immune deficiency and DNA repair
Authors: Schwarz Klaus , Ma Yunmei , Pannicke Ulrich , Lieber Michael R.
Published: October 2003

Human severe combined immune deficiency (SCID) is the most serious inherited immunological deficit. Recent work has revealed defects in the predominant pathway for double‐strand break repair called nonhomologous DNA end joining, or NHEJ. Progress in the biochemistry and genetics of NHEJ and of human SCID has proven to be synergistic between these two fields in a manner that covers the range from biochemical etiology to considerations about possible gene therapy for the B− SCID patients. BioEssays 25:1061–1070, 2003. © 2003 Wiley Periodicals, Inc.
Growing and shaping the vascular tree: multiple roles for VEGF
Authors: Ruhrberg Christiana
Published: October 2003

Vascular Endothelial Growth Factor (VEGF) is the most potent and ubiquitous vascular growth factor known to date. Yet, prior to its description as a secreted mitogen for endothelial cells, it was identified as a vascular permeability factor. These seemingly disparate avenues of discovery highlight VEGF's ability to control many distinct aspects of endothelial cell behaviour, including proliferation, migration, specialisation and survival. The versatility of VEGF as a patterning molecule is likely linked to its association with various signalling receptor complexes, but also its expression in several isoforms with a differential affinity for heparan sulfate proteoglycans in the extracellular matrix. In contrast to the absolute requirement for all known VEGF receptors, the presence of only a single VEGF isoform is sufficient for vascular development. However, the isoforms serve as exquisite tools for the fine patterning of growing vessel networks during embryogenesis and in postnatal life. BioEssays 25:1052–1060, 2003. © 2003 Wiley Periodicals, Inc.
Chronic activation of ERK and neurodegenerative diseases
Authors: Colucci‐D'Amato Luca , Perrone‐Capano Carla , di Porzio Umberto
Published: October 2003

The extracellular‐signal regulated kinases 1/2 (ERK or ERKs) are involved in the regulation of important neuronal functions, including neuronal plasticity in normal and pathological conditions. We present findings that support the notion that the kinetics and localization of ERK are intrinsically linked, in that the duration of ERK activation dictates its subcellular compartmentalization and/or trafficking. The latter, in turn, dictates whether ERK‐expressing cells would enter a program of cell death, survival or differentiation. We summarize experimental data showing that chronic activation of ERK plays a role in the mechanisms that trigger neurodegeneration. We also discuss how MKPs, members of the subclass of dual specificity phosphatases, might be the link between ERK kinetics and its subcellular localization. BioEssays 25:1085–1095, 2003. © 2003 Wiley Periodicals, Inc.
Crystal structure of achiral nonapeptide Boc–(Aib–ΔzPhe)4–Aib–OMe at atomic resolution: Evidence for a 310‐helix
Authors: Inai Yoshihito , Oshikawa Tatsuo , Yamashita Mitsuji , Tagawa Kenichi , Hirabayashi Tadamichi
Published: October 2003

An x‐ray crystallographic analysis was carried out for Boc–(Aib–ΔZPhe)4–Aib–OMe (1: Boc = t‐butoxycarbonyl; Aib = α‐aminoisobutyric acid; ΔZPhe = Z‐α,β‐didehydrophenylalanine) to provide the precise conformational parameters of the octapeptide segment –(Aib–ΔZPhe)4–. Peptide 1 adopted a typical 310‐helical conformation characterized by 〈ϕ〉 = ±55.8° (50°–65°), 〈ψ〉 = ±26.7° (15°–45°), and 〈ω〉 = ±179.5° (168°–188°) for the average values of the –(Aib–ΔZPhe)4– segment (the range of the eight values). The 310‐helix contains 3.1 residues per turn, being close to the “perfect 310‐helix” characterized by 3.0 residues per turn. NMR and Fourier transform infrared (FTIR) spectroscopy revealed that the 310‐helical conformation at the atomic resolution is essentially maintained in solution. Energy minimization of peptide 1 by semiempirical molecular orbital calculation converged to a 310‐helical conformation similar to the x‐ray crystallographic 310‐helix. The preference for a 310‐helix in the –(Aib–ΔZPhe)4– segment is ascribed to strong inducers of the 310‐helix inherent in Aib and ΔZPhe residues—in particular, the Aib residues tend to stabilize a 310‐helix more effectively. Therefore, the –(Aib–ΔZPhe)4– segment is useful to rationally design an optically inactive 310‐helical backbone, which will be of great importance to provide novel insights into noncovalent and covalent chiral interactions of a helical peptide with a chiral molecule. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 310–322, 2003
Structural transitions involved in a novel amyloid‐like β‐sheet assemblage of tripeptide derivatives
Authors: Ganesh S. , Jayakumar R.
Published: October 2003

Self‐assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5×10−4M). However, at high concentration (2×10−3M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far‐uv circular dichroism and Fourier transform infrared (FT‐IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of β‐sheet, retaining a α‐helical contribution. Peptide A showed a conformational transition resulting in a β‐sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel β‐sheet conformation, whereas peptide A takes a parallel β‐sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 336–345, 2003
Adsorption of actin at the air‐water interface: A monolayer study
Authors: Gicquaud C. , Chauvet J.‐P. , Grenier G. , Tancrède P. , Coulombe G.
Published: October 2003

The intrinsic surface activity of the contractile protein actin has been determined from surface tension measurements using the Wilhelmy hanging‐plate method. Actin, a very soluble protein, moves from the subphase to the air‐water interface to make a film. In the absence of magnesium, actin is monomeric and is known as G‐actin. During the compression the monomers change their conformation or orientation at the interface and they are then pushed reversibly into the subphase upon further compression. No collapse occurs. Actin monomers in the presence of magnesium become activated; at concentrations greater than some critical value, actin polymerizes to form filaments of F‐actin. The actin filaments have a higher surface activity than the actin monomers either because they are more hydrophobic or because F‐actin, a rigid polymer, is much more efficient at creating excluded volume. The actin filaments then form a rigid film at the interface that collapses when the surface area is decreased. At less than the critical concentration, the actin monomers are present in the subphase in their activated form. However, their concentration increases at the interface during film compression until the critical concentration is reached. The surface pressure isotherm in this case has the characteristics of a G‐actin film at the beginning of the compression and of an F‐actin film at the end of the compression process. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 289–296, 2003
Small‐angle X‐ray scattering studies of metastable intermediates of β‐lactoglobulin isolated after heat‐induced aggregation
Authors: Carrotta Rita , Arleth Lise , Pedersen Jan Skov , Bauer Rogert
Published: October 2003

Small‐angle x‐ray scattering was used for studying intermediate species, isolated after heat‐induced aggregation of the A variant of bovine β‐lactoglobulin. The intermediates were separated in two fractions, the heated metastable dimer and heated metastable oligomers larger than the dimer. The pair distance distribution functions for the two intermediate fractions as well as for the native protein have been obtained by indirect Fourier transformation. In addition, the scattering intensity data for samples of the native protein at different concentrations were fitted using a combination of monomer and dimer form factors, which provides an estimate of the amount of monomer in solutions. By subtracting the contribution from the monomer, the scattering intensity from the dimer of the native protein can be determined and compared with the results for the metastable dimer. An ellipsoidal model was used to fit the data for the metastable dimer, and for comparison the same analysis was performed on the dimer of the native protein. The results show that the metastable dimer is more elongated than the dimer of the native protein and it occupies a volume 1.4‐fold larger, in agreement with a more loose, partially unfolded conformation. The same ellipsoidal model was used to analyze the data for the fraction of larger metastable oligomers. In this case, an even more elongated ellipsoid was obtained, suggesting a linear association of monomers in the oligomers. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 377–390, 2003
Brownian dynamics simulations of ion atmospheres around polyalanine and B‐DNA: Effects of biomolecular dielectric
Authors: Cerutti David S. , Wong Chung F. , McCammon J. Andrew
Published: October 2003

We have extended an earlier Brownian dynamics simulation algorithm for simulating the structural dynamics of ions around biomolecules to accommodate dielectric inhomogeneity. The electrostatic environment of a biomolecule immersed in water was obtained by numerically solving the Poisson equation with the biomolecule treated as a low dielectric region and the solvent treated as a high dielectric region. Instead of using the mean‐field type approximations of ion interactions as in the Poisson–Boltzmann model, the ions were treated explicitly by allowing them to evolve dynamically under the electrostatic field of the biomolecule. This model thus accounts for ion–ion correlations and the finite‐size effects of the ions. For a 13‐residue α‐helical polyalanine and a 12‐base‐pair bp B‐form DNA, we found that the choice of the dielectric constant of the biomolecule has much larger effects on the mean ionic structure around the biomolecule than on the fluctuational and dynamical properties of the ions surrounding the biomolecule. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 391–402, 2003
Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility
Authors: Jia Hongfei , Zhu Guangyu , Wang Ping
Published: October 2003

Nanoparticles provide an ideal remedy to the usually contradictory issues encountered in the optimization of immobilized enzymes: minimum diffusional limitation, maximum surface area per unit mass, and high effective enzyme loading. In addition to the promising performance features, the unique solution behaviors of the nanoparticles also point to a transitional region between the heterogeneous (with immobilized enzymes) and homogeneous (with soluble free enzymes) catalysis. The particle mobility, which is related to particle size and solution viscosity through Stokes‐Einstein equation, may impact the reaction kinetics according to the collision theory. The mobility‐activity relationship was examined through experimental studies and theoretical modeling in the present work. Polystyrene particles with diameters ranging from 110–1000 nm were prepared. A model enzyme, α‐chymotrypsin, was covalently attached to the nanoparticles up to 6.6 wt%. The collision theory model was found feasible in correlating the catalytic activities of particles to particle size and solution viscosity. Changes in the size of particles and the viscosity of reaction media, which all affect the mobility of the enzyme catalyst, evidently altered the intrinsic activity of the particle‐attached enzyme. Compared to KM, kcat appeared to be less sensitive to particle size and viscosity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 406–414, 2003.
Measuring local flow velocities and biofilm structure in biofilm systems with Magnetic Resonance Imaging (MRI)
Authors: Manz Bertram , Volke Frank , Goll Danile , Horn Harald
Published: October 2003

The characterization of substrate transport in the bulk phase and in the biofilm matrix is one of the problems which has to be solved for the verification of biofilm models. Additionally, the surface structure of biofilms has to be described with appropriate parameters. Magnetic resonance imaging (MRI) is one of the promising methods for the investigation of transport phenomena and structure in biofilm systems. The MRI technique allows the noninvasive determination of flow velocities and biofilm structures with a high resolution on the sub‐millimeter scale. The presented investigations were carried out for defined heterotrophic biofilms which were cultivated in a tube reactor at a Reynolds number of 2000 and 8000 and a substrate load of 6 and 4 g/m2d glucose. Magnetic resonance imaging provides both structure data of the biofilm surface and flow velocities in the bulk phase and at the bulk/biofilm interface. It is shown that the surface roughness of the biofilms can be determined in one experiment for the complete cross section of the test tubes both under flow and stagnant conditions. Furthermore, the local shear stress was calculated from the measured velocity profiles. In the investigated biofilm systems the local shear stress at the biofilm surface was up to 3 times higher compared to the mean wall shear stress calculated on the base of the mean flow velocity. © 2003, Wiley Periodicals, Inc. Biotechnol Bioeng 84: 424–432, 2003.
Enzymatic activity and stability of d‐fructose dehydrogenase and sarcosine dehydrogenase immobilizd onto giant vesicles
Authors: Kato Keiichi , Walde Peter , Mitsui Hirokazu , Higashi Nobuyasu
Published: October 2003

Stable vesicles with diameters between about 1 and 10 μm were prepared by a particular emulsification technology that involved the use of the surfactants Span 80 and Tween 80 and the phospholipid lecithin (phosphatidylcholine from soybeans). Two membrane enzymes, d‐fructose dehydrogenase from Gluconobacter sp. (FDH) and sarcosine dehydrogenase from Pseudomonas putida (SDH), were for the first time immobilized onto the bilayer membranes of these type of vesicles; and the catalytic activity and enzymatic stability were measured and compared with the enzymes in a vesicle‐free solution. The enzyme activity as well as stability considerably increased upon immobilization. In particular, immobilized FDH at 25°C was stable for at least 20 days, while the activity of the free enzyme dropped to about 20% of its initial value during the same period of time.

In contrast to FDH and SDH, immobilization of sorbitol dehydrogenase from Gluconobacter suboxydans (SODH) was not successful, as no improved activity or stability could be obtained. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 415–423, 2003.
One‐pot enzymatic production of dTDP‐4‐Keto‐6‐Deoxy‐d‐glucose from dTMP and glucose‐1‐phosphate
Authors: Oh Jongmin , Lee Sun‐Gu , Kim Byung‐Gee , Sohng Jae Kyung , Liou Kwangkyoung , Lee Hei Chan
Published: October 2003

An enzymatic production method for dTDP‐4‐keto‐6‐deoxy‐d‐glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose‐1‐phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP‐glucose synthase, and dTDP‐d‐glucose 4,6‐dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP‐4‐keto‐6‐deoxy‐d‐glucose was synthesized by using the enzyme extracts in one‐pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose‐1‐phosphate concentrations were optimized. About 95% conversion yield of dTDP‐4‐keto‐6‐deoxy‐d‐glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose‐1‐phosphate, and 20 mM MgCl2. The rate‐limiting step in this multiple enzyme reaction system was the dTDP‐glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP‐4‐keto‐6‐deoxy‐d‐glucose was obtained in an overall yield of 81% after two‐step purification, i.e., anion exchange chromatography and gel filtration. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 452–458, 2003.
High‐level expression of full‐length antibodies using trans‐complementing expression vectors
Authors: Bianchi Allison A. , McGrew Jeffrey T.
Published: October 2003

Recombinant antibodies are increasingly used as therapeutics for a wide variety of diseases. Generation of cell lines expressing high levels of recombinant antibody typically requires labor‐intensive cloning and screening steps. We describe a mammalian expression system for the high‐level production of full‐length antibody molecules. It has been shown that the dihydrofolate reductase (DHFR) selectable marker can be divided into two fragments that, with the aid of a leucine zipper, can re‐associate to form an active molecule. Using bicistronic vectors, we linked the expression of each antibody chain to the expression of a DHFR fragment. Survival in selective media requires expression of both DHFR fragments that, by virtue of these vectors, also selects for the expression of both antibody chains. Initial pools produced 5 µg of Ab/106 cells/d (qP = µg/106 cells/d). Expression of each antibody chain in conjunction with a portion of DHFR also leads to concurrent amplification of both antibody chains in the presence of methotrexate, a DHFR inhibitor, and results in a two‐ to fivefold increase in antibody production with basal qPs ranging from 10–25 ug/106 cells/d. Shake‐flask cultures of amplified pools produced up to 600 mg/L of antibody in 7 days. This system allows for rapid generation of antibodies without cloning and greatly simplifies selection of cell lines for the production of potential antibody therapeutics. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 439–444, 2003.
Effect of mixing rate on β‐carotene production and extraction by dunaliella salina in two‐phase bioreactors
Authors: Hejazi M. A. , Andrysiewicz E. , Tramper J. , Wijffels R. H.
Published: October 2003

β‐Carotene has many applications in the food, cosmetic, and pharmaceutical industries; Dunaliella salina is currently the main source for natural β‐carotene. We have investigated the effect of mixing rate and whether it leads to the facilitated release of β‐carotene from the cells of Dunaliella salina in two‐phase bioreactors. Three pairs of bioreactors were inoculated at the same time, operated at 100, 150, and 170 rounds per minute, respectively, and illuminated with a light intensity of 700 μmol m−2 s−1. Each pair consisted of one bioreactor containing only aqueous phase for the blank and one containing the water phase together with dodecane, which is biocompatible with the cells. Comparison of the viability and growth of the cells grown under different agitation rates shows that 170 rpm and 150 rpm are just as good as 100 rpm. The presence and absence of the organic phase also has no influence on the viability and growth of the cells. In contrast to the growth rate, the extraction rate of β‐carotene is influenced by the stirrer speed. The extraction rate increases at a higher stirring rate. The effectiveness of extraction with respect to power input is comparable for all the applied mixing rates, even though it is slightly lower for 100 rpm than the others. The chlorophyll concentration in the organic phase remained very low during the experiment, although at higher mixing rates, chlorophyll impurity increased up to 3% (w/w) of the total extracted pigments. At 170 rpm carotenoid and chlorophyll undergo the highest extraction rate for both pigments—0.5% of the chlorophyll and 6% of the carotenoid is extracted. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 591–596, 2003.
Higher expression of fab antibody fragments in a CHO cell line at reduced temperature
Authors: Schatz Simone M. , Kerschbaumer Randolf J. , Gerstenbauer Gabriele , Kral Martina , Dorner Friedrich , Scheiflinger Friedrich
Published: October 2003

A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two‐stage production process. Cells were first grown to 90% confluence at 37°C in a proliferation phase, followed by a production phase at either 37°C or 28°C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28°C resulted in a 14‐fold increase in product yield when cells were cultivated in serum‐containing medium, and in a 38‐fold increase in product yield when serum‐free medium was applied. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 433–438, 2003.
Optical processing of bacterial libraries for directed evolution
Authors: Scruggs Allan Wallace , Woodbury Neal W.
Published: October 2003

Selection of phenotypically distinct bacterial colonies on a Petri dish is typically performed by one of two methods: chemical or mechanical. Chemical methods (e.g., antibiotic selection) rely on inherent growth advantages of the unique phenotypes desired and thus have limited applicability. Mechanical methods are generally slow and require relatively large colonies (typically hundreds of colonies per plate). Here the use of imaged light to select bacterial colonies is explored, employing either photodynamic therapy agents or a ferrochelatase mutation in combination with porphyrin precursors to sensitize the bacteria to light and a computer‐controlled light projection system to illuminate some bacterial colonies while leaving others in the dark. A CCD camera was used to distinguish between bacteria expressing green fluorescent protein (GFP) from nonfluorescent colonies. The fluorescence image from the camera was then used to create a virtual masking image for photoselection. Using a simple commercial projector it was possible to confer a 56‐fold selective advantage to colonies expressing GFP. This represents a potentially powerful tool in directed evolution experiments using large libraries. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 445–451, 2003.
Development of a fed‐batch fermentation process to overproduce phosphoenolpyruvate carboxykinase using an expression vector with promoter and plasmid copy number controllable by heat
Authors: Chao Yun‐Peng , Chern Jong‐Tzer , Lin Wei Shing , Wang Zei Wen
Published: October 2003

To effectively achieve tight regulation and high‐level expression of cloned genes, a novel expression plasmid has been developed to contain the promoter and allow the plasmid copy number to be controlled by heat. The feasibility of the plasmid was tested by overproducing the pck gene product (Pck), a protein responsible for cell growth on gluconeogenic carbons and with potential toxicity. By fusing the pck gene with the promoter on the plasmid, the Escherichia coli strain harboring the composite vector was shown to produce various amounts of Pck in response to different degrees of heat shock. With the use of a 30°→41°C stepwise upshift, the shake‐flask culture of recombinant cells enabled production of maximal Pck in soluble form accounting for 20% of total cell protein. In sharp contrast, Pck production was undetectable in the uninduced cell, and this was further confirmed by the failed growth of strain JCL1305, defective in the essential genes for gluconeogenesis, carrying the composite vector on succinate at 30°C. By exploiting the fed‐batch fermentation approach, the recombinant cell batch initially kept at 30°C in a lab‐scale fermentor was exposed to 41°C for 2 h at the batch fermentation stage, followed by a reduction in temperature to 37°C throughout the remainder of the culturing process. Consequently, this resulted in Pck production equivalent to 15% of total cell protein. The total Pck yield thus calculated was amplified 1880‐fold over that obtained at the shake‐flask scale. Overall, there is great promise for this expression system due to its tight control, high production, simple thermomodulation, and feasible scale‐up of recombinant proteins. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 459–466, 2003.
Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in escherichia coli
Authors: Seo Jeong Hyun , Li Lin , Yeo Joo Sang , Cha Hyung Joon
Published: October 2003

Baculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system. We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP). The polyhedrin fusion protein (∼58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein. The E. coli expressing polyhedrin–GFP fusion protein showed higher cell growth (∼1.8‐fold) and higher GFP yield (∼3.5‐fold) than the strain expressing soluble single GFP. Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut. In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by α‐chymotrypsin, a protease that has similar properties to insect midgut polyhedra‐associated alkaline proteases. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E. coli expression systems. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 467–473, 2003.
Supplementation‐dependent differences in the rates of embryonic stem cell self‐renewal, differentiation, and apoptosis
Authors: Viswanathan Sowmya , Benatar Tania , Mileikovsky Maria , Lauffenburger Douglas A. , Nagy Andras , Zandstra Peter W.
Published: October 2003

Although it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4‐GFP‐positive (ES) and ‐negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose‐dependent survival and proliferation advantage, thus enabling the long‐term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor‐4 (FGF‐4), or insulin‐like growth factor‐1 (IGF‐1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 505–517, 2003.
Degradation of xenobiotics in a partitioning bioreactor in which the partitioning phase is a polymer
Authors: Amsden Brian G. , Bochanysz Justyna , Daugulis Andrew J.
Published: October 2003

Two‐phase partitioning bioreactors (TPPBs) are characterized by a cell‐containing aqueous phase and a second immiscible phase that contains toxic and/or hydrophobic substrates that partition to the cells at subinhibitory levels in response to the metabolic demand of the organisms. To date, the delivery phase in TPPBs has been a hydrophobic solvent that traditionally needed to possess a variety of important properties including biocompatibility, nonbioavailability, low volatility, and low cost, among others. In the present work we have shown that the organic solvent phase can be replaced by inexpensive polymer beads that function in a similar fashion as organic solvents, delivering a toxic substrate to cells based on equilibrium considerations. Specifically, 3.4 mm diameter beads of poly(ethylene‐co‐vinyl acetate) (EVA) were used to reduce the aqueous concentration of phenol in a bioreactor from toxic levels ( ∼2,000 mg/L) to subinhibitory levels (∼750 mg/L), after which Pseudomonas putida ATCC 11172 was added to the system and allowed to consume the total phenol loading. Thus, the beads absorbed the toxic substrate and released it to the cells on demand. The EVA beads, which could be reused, were able to absorb 14 mg phenol/g EVA. This work has opened the possibility of using widely mixed cultures in TPPB systems without concern for degradation of the delivery material and without concern of contamination. © 2003 Wiley Periodicals. Biotechnol Bioeng 84: 399–305, 2003.
Further studies related to the scale‐up of high cell density escherichia coli fed‐batch fermentations:
Authors: Onyeaka Helen , Nienow Alvin W. , Hewitt Christopher J.
Published: October 2003

In this work, we report on the further development of the scale‐down, two‐compartment (STR + PFR) experimental simulation model. For the first time, the effect on high cell density Escherichia coli fed‐batch fermentations of a changing microenvironment with respect to all three of the major spatial heterogeneities that may be associated with large‐scale processing (pH, glucose, and dissolved oxygen concentration) were studied simultaneously. To achieve this, we used traditional microbiological analyses as well as multiparameter flow cytometry to monitor cell physiological response at the individual cell level. It was demonstrated that for E. coli W3110 under such conditions in a 20 m3 industrial fed‐batch fermentation, the biomass yield is lower and final cell viability is higher than those found in the equivalent well‐mixed, 5L laboratory scale case. However, by using a combination of the well‐mixed 5L stirred tank reactor (STR) with a suitable plug flow reactor (PFR) to mimic the changing microenvironment at the large scale, very similar results to those in the 20 m3 reactor may be obtained. The similarity is greatest when the PFR is operated with a mean residence time of 50 sec with a low level of dO2 and a high glucose concentration with either a pH of 7 throughout the two reactors or with pH controlled at 7 in the STR by addition into the PFR where the pH is > 7. © 2003 Wiley Periodicals. Biotechnol Bioeng 84: 474–484, 2003.