bluuebunny/crossref_metadata_2025 huggingface dataset:
NMR identification of left‐handed polyproline type II helices
Authors: Lam Sik Lok , Hsu Victor L.
Published: May 2003

NMR characteristics of a model left‐handed 31‐helical peptide are reported in this study. With temperature and sequence corrections on the predicted random coil 15N chemical shifts, a significant 15N chemical shift deviation is observed for the model 31 peptide. The 15N chemical shift differences also correlate well with the molar ellipticities (at 220 nm) of the CD spectra at different temperatures, indicating that the 15N chemical shift is a sensitive probe for 31‐helices. The average 3JHNα and 1JCαHα values of the model peptide are determined to be 6.5 and 142.6 Hz, respectively, which are consistent with the values calculated from the geometry of 31‐helices. With careful measurements of amide 15N chemical shifts and incorporating temperature and sequence effect corrections, the 15N chemical shifts can be used together with 3JHNα and 1JCαHα to differentiate 31‐helices from random coils with high confidence. Based on the observed NMR characteristics, a strategy is developed for probing left‐handed 31‐helical structures from other secondary structures. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 270–281, 2003
Local and long‐range structural effects caused by the removal of the N‐terminal polypeptide fragment from immunoglobulin L chain λ
Authors: Król Marcin , Roterman Irena , Piekarska Barbara , Konieczny Leszek , Rybarska Janina , Stopa Barbara
Published: May 2003

The role of the N‐terminal polypeptide fragment of the immunoglobulin l‐chain in V domain packing stability, and the flexibility of the whole chain was approached by molecular dynamics simulation. The observations were supported by experimental analysis. The N‐terminal polypeptide fragment appeared to be the low‐stability packing element in the V domain. At moderately elevated temperature it may be replaced at its packing locus by Congo red and then removed by proteolysis. After removal of Congo red by adsorption to (diethylamino)ethyl (DEAE) cellulose, the stability of complete L chain and of L chain devoid of the N‐terminal polypeptide fragment were compared. The results indicated that the N‐terminal polypeptide fragment plays an essential role in the stability of the V domain. Its removal makes the domain accessible for ANS and Congo red dye binding without heating. The decreased domain stability was registered in particular as increased root mean square (RMS) fluctuation and higher susceptibility to proteolytic attack. The long‐range effect was most clearly manifested at 340 K as independent V and C domain fluctuation in the l‐chain devoid of the N‐terminal polypeptide fragment. This is likely due to the lack of direct connections between the N‐ and C‐termini of the V domain polypeptide. In a complete V domain the connection involves residues 8–12 and 106–110 in particular. Partial or complete disruption of this connection increases the freedom of V domain rotation, while its increased cohesion strengthens the coupling of the V and C domains, making the whole L chain less flexible. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 189–200, 2003
The NMR‐derived conformation of neuropeptide AF, an orphan G‐protein coupled receptor peptide
Authors: Miskolzie Mark , Kotovych George
Published: May 2003

The tertiary structure of the pain modulating and anti‐opiate neuropeptide, human neuropeptide AF (NPAF) (the sequence is AGEGLNSQFWSLAAPQRF–NH2), was determined by 1H‐NMR. The structure of NPAF was determined in two solvent systems, namely 50%/50% trifluoroethanol‐d3/H2O (TFE/H2O) and in the cell membrane mimetic micelle, sodium dodecylsulfate‐d25 (SDS). The receptor for NPAF is an orphan G‐protein coupled receptor, and the micellar SDS solvent system was used to emulate the cell membrane surface in line with the Cell Membrane Compartments Theory proposed by R. Schwyzer (Biopolymers, 1995, Vol. 37, pp. 5–16). In both solvent systems, NPAF was found to be primarily α‐helical within the central portion of the molecule, from Asn6 to Ala14. The N‐terminus was random in both solvent systems. In the SDS solution, the C‐terminal tetrapeptide was structured and formed a type I β‐turn, whereas in TFE/H2O it was unstructured, showing the importance of the C‐terminal tetrapeptide in receptor recognition. NPAF was found to associate with SDS, and was shown to be near the surface of the micelle by spin label studies with 5‐doxyl‐stearic acid. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 201–215, 2003
Molecular dynamics simulation studies of cytochrome b5 from outer mitochondrial and microsomal membrane
Authors: Lee Kyung‐Hoon , Kuczera Krzysztof
Published: May 2003

Two forms of cytochrome b5 have been identified, associated with the outer membrane of liver mitochondria (OM cyt b5) and with the membrane of the endoplasmic reticulum (microsomal, Mc cyt b5). These proteins have very similar structures, but differ significantly in physical properties, with the OM cyt b5 exhibiting a more negative reduction potential, higher stability, and stronger interactions with the heme. We perform molecular dynamics simulations to probe the structures and fluctuations of the two proteins in solution, to help explain the observed physical differences. We find that the structures of the two proteins, highly similar in the crystal, differ in position of a surface loop involving residues 49–51 in solution. Hydrophobic residues Ala‐18, Ile‐32, Leu‐36, and Leu‐47 tend to cluster together on the surface of rat OM cyt b5, blocking water access to the protein interior. In bovine Mc cyt b5, two of these positions, Ser‐18 and Arg‐47, are occupied by hydrophilic residues. This leads to breaking the hydrophobic cluster and allowing the protein to occupy a more open conformation. A measure of this structural transition is the opening of a cleft on the protein surface, which is 5 Å wider in the OM cyt b5 simulation compared to the Mc form. The OM protein also appears to have a more compact hydrophobic core in its β‐sheet region. These effects may be used to explain observed stability differences between the two proteins. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 260–269, 2003
Zinc binding in peptide models of angiotensin‐I converting enzyme active sites studied through 1H‐NMR and chemical shift perturbation mapping
Authors: Galanis Athanassios S. , Spyroulias Georgios A. , Pierattelli Roberta , Tzakos Andreas , Troganis Anastassios , Gerothanassis Ioannis P. , Pairas George , Manessi‐Zoupa Evy , Cordopatis Paul
Published: May 2003

We report the design and synthesis through solid phase 9‐flourenylmethoxycarbonyl (Fmoc) chemistry of the two angiotensin‐I converting enzyme active sites possessing the general sequence HEMGHX23EAIGDX3. Their zinc‐binding properties were monitored in solution through high‐resolution 1H‐NMR. The obtained data were analyzed in terms of chemical shift differences. The results indicate that zinc binds to the HEMGH and the EAIGD characteristic motifs, and suggest possible coordination modes of zinc in the native enzyme. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 244–252, 2003
Synthetic peptide vaccine and antibody therapeutic development: Prevention and treatment of Pseudomonas aeruginosa
Authors: Cachia Paul J. , Hodges Robert S.
Published: May 2003

Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti‐adhesin vaccine and a related therapeutic monoclonal antibody (cross‐reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell‐surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti‐adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 141–168, 2003
Grb2 SH2 domain‐binding peptide analogs as potential anticancer agents
Authors: Lung Feng‐Di T. , Tsai Jya‐Yin
Published: May 2003

The growth factor receptor‐bound protein 2 (Grb2) plays an important role in the Ras signaling pathway. Several proteins were found to be overexpressed by oncogenes in the Ras signaling pathway, rendering Grb2 a potential target for the design of antitumor agents. Blocking the interaction between the phosphotyrosine‐containing activated receptor and the Src‐homology 2 (SH2) domain of Grb2 thus constitutes an important strategy for the development of potential anticancer agents. X‐ray, NMR structural investigations, and molecular modeling studies have provided the target structure of Grb2 SH2 domain—alone or complexed with a phosphotyrosine‐containing peptide—which is useful for the structure‐based design of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain. We review here the variety of approaches to Grb2 SH2 pepide inhibitors developed with the aim of interrupting Grb2 recognition. Inhibitory effects of peptide analogs on the Grb2 SH2 domain and their binding affinities for Grb2 SH2 were determined by ELISA, cell‐based assays, or Surface Plasman Resonance (SPR) technology. Results of theses studies provide important information for further modifications of lead peptides, and should lead to the discovery of potent peptides as anticancer agents. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 132–140, 2003
Oligopeptide inhibition of class I ribonucleotide reductases
Authors: Cooperman Barry S.
Published: May 2003

Class I ribonucleotide reductases (RRs), which are well‐recognized targets for cancer chemotherapeutic and antiviral agents, are composed of two different subunits, R1 and R2, and are inhibited by oligopeptides corresponding to the C‐terminus of R2, which compete with R2 for binding to R1. These peptides specifically inhibit the RRs from which they are derived, and closely homologous RRs, but do not inhibit less homologous RRs. Here we review results obtained for oligopeptide inhibition of RRs from several sources, including related x‐ray, NMR, and modeling results. The most extensive studies have been performed on herpes simplex virus‐RR (HSV‐RR) and mammalian‐RR (mRR). A common model fits the data obtained for both enzymes, in which the C‐terminal residue of the oligopeptide (Leu for HSV‐RR, Phe for mRR) binds with high specificity to a narrow and deep hydrophobic subsite, and two or more hydrophobic groups at the N‐terminal portion of the peptide bind to a broad and shallow second hydrophobic subsite. The studies have led to the development of highly potent and specific inhibitors of HSV‐RR and promising inhibitors of mRR, and indicate possible directions for the development of inhibitors of bacterial and fungal RRs. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 117–131, 2003
Successful identification of novel agents to control infectious diseases from screening mixture‐based peptide combinatorial libraries in complex cell‐based bioassays
Authors: Boggiano César , Reixach Natàlia , Pinilla Clemencia , Blondelle Sylvie E.
Published: May 2003

Mixture‐based peptide synthetic combinatorial libraries (SCLs) represent a valuable source for the development of novel agents to control infectious diseases. Indeed, a number of studies have now proven the ability of identifying active peptides from libraries composed of thousands to millions of peptides in cell‐based biosystems of varying complexity. Furthermore, progressing knowledge on the importance of endogenous peptides in various immune responses lead to a regain in importance for peptides as potential therapeutic agents. This article is aimed at providing recent studies in our laboratory for the development of antimicrobial or antiviral peptides derived from mixture‐based SCLs using cell‐based assays, as well as a short review of the importance of such peptides in the control of infectious diseases. Furthermore, the use of positional scanning (PS) SCL‐based biometrical analyses for the identification of native optimal epitopes specific to HIV‐1 proteins is also presented. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 103–116, 2003
Dmt and opioid peptides: A potent alliance
Authors: Bryant Sharon D. , Jinsmaa Yunden , Salvadori Severo , Okada Yoshio , Lazarus Lawrence H.
Published: May 2003

The introduction of the Dmt (2′,6′‐dimethyl‐L‐tyrosine)–Tic pharmacophore into the design of opioid ligands produced an extraordinary family of potent δ‐opioid receptor antagonists and heralded a new phase in opioid research. First reviewed extensively in 1998, the incorporation of Dmt into a diverse group of opioid molecules stimulated the opioid field leading to the development of unique analogues with remarkable properties. This overview will document the crucial role played by this residue in the proliferation of opioid peptides with high receptor affinity (Ki equal to or less than 1 nM) and potent bioactivity. The discussion will include the metamorphosis between δ‐opioid receptor antagonists to δ‐agonists based solely on subtle structural changes at the C‐terminal region of the Dmt–Tic pharmacophore as well as their behavior in vivo. Dmt may be considered promiscuous due to the acquisition of potent μ‐agonism by dermorphin and endomorphin derivatives as well as by a unique class of opioidmimetics containing two Dmt residues separated by alkyl or pyrazinone linkers. Structural studies on the Dmt–Tic compounds were enhanced tremendously by x‐ray diffraction data for three potent and biologically diverse Dmt–Tic opioidmimetics that led to the development of pharmacophores for both δ‐opioid receptor agonists and antagonists. Molecular modeling studies of other unique Dmt opioid analogues illuminated structural differences between δ‐ and μ‐receptor ligand interactions. The future of these compounds as therapeutic applications for various medical syndromes including the control of cancer‐associated pain is only a matter of time and perseverance. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71:86–102, 2003
Sequence‐dependent solution structure and motions of 13 TATA/TBP (TATA‐box binding protein) complexes
Authors: Strahs Daniel , Barash Danny , Qian Xiaoliang , Schlick Tamar
Published: May 2003

The TATA element is a well‐known example of a DNA promoter sequence recognized by the TATA box binding protein (TBP) through its intrinsic motion and deformability. Although TBP recognizes the TATA element octamer unusually (through the minor groove, which lacks the distinctive features of the major groove), single base‐pair replacements alter transcriptional activity. Recent crystallographic experiments have suggested that TATA/TBP complexes differing by a single base pair retain substantial structural similarity despite their functional differences in activating transcription. To investigate the subtle role of sequence‐dependent motion within the TATA element and certain aspects of its effect on assembly of the transcriptional complex, we examine 5‐ns dynamics trajectories of 13 variant TATA/TBP complexes differing from each other by a single base pair. They include the wild‐type (WT) adenovirus 2 major late promoter (AdMLP) TATA element, TATAAAAG (the octamer specifies positions −31 to −24 with respect to the transcription initiation site), and the variants A31 (i.e., AATAAAAG), T30, A29, C29, G28, T28, T27, G26, T26, C25, T25, and T24. Our simulated TATA/TBP complexes develop sequence‐dependent structure and motion trends that may lead to favorable orientations for high‐activity variants (with respect to binding TFIIA, TFIIB, and other transcription factors), while conversely, accelerate dissociation of low‐activity TATA/TBP complexes. The motions that promote favorable geometries for preinitiation complexes include small rotations between TBP's N‐ and C‐terminal domains, sense strand DNA backbone “slithering,” and rotations in TBP's H2 and H2′ helices. Low‐activity variants tend to translate the H1 and H1′ helices and withdraw the intercalating phenylalanines. These cumulative DNA and protein motions lead to a spatial spread of complex orientations up to 4 Å; this is associated with an overall bend of the variant TATA/TBP complexes that spans 93° to 110° (107° for the crystal reference). Taken together, our analyses imply larger differences when these local structural and bending changes are extended to longer DNA (upstream and downstream) and suggest that specific local TATA/TBP motions (e.g., shifts in TBP helices and TATA bases and backbone) play a role in modulating the formation and maintenance of the transcription initiation complex. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 216–243, 2003
Homology model of the CB1 cannabinoid receptor: Sites critical for nonclassical cannabinoid agonist interaction
Authors: Shim Joong‐Youn , Welsh William J. , Howlett Allyn C.
Published: May 2003

Association of cannabimimetic compounds such as cannabinoids, aminoalkylindoles (AAIs), and arachidonylethanolamide (anandamide) with the brain cannabinoid (CB1) receptor activates G‐proteins and relays signals to regulate neuronal functions. A CB1 receptor homology model was constructed using the published x‐ray crystal structure of bovine rhodopsin (Palczewski et al., Science,2000, Vol. 289, pp. 739–745) in the conformation most likely to represent the “high‐affinity” state for agonist binding to G‐protein coupled receptors (GPCRs). A molecular docking approach that combined Monte Carlo and molecular dynamics simulations was used to identify the putative binding conformations of nonclassical cannabinoid agonists, including AC‐bicyclic CP47497 and CP55940, and ACD‐tricyclic CP55244. Placement of these ligands was based upon the assumption of a critical hydrogen bond between the A‐ring OH and the side chain N of Lys192 in transmembrane helix 3. We evaluated two alternative binding conformations, C3‐in and C3‐out, denoting the directionality of the ligand C3 side chain within the receptor with respect to the inside or the outside of the cell. Assuming both the C3‐in or C3‐out conformation, the calculated ligand–receptor binding energy (ΔEbind) was correlated with the experimentally observed binding affinity (Ki) for a series of nonclassical cannabinoid agonists. The C3‐in conformation was marginally better than the alternative C3‐out conformation in predicting the rank order of the tested nonclassical cannabinoid analogs. Adopting the C3‐in conformation due to the greater number of receptor interactions with known pharmacophoric elements of the ligand, key residues were identified comprising the presumed hydrophobic pocket that interacts with the C3 side chain of cannabinoid agonists. Key hydrogen bonds would form between both K3.28(192) and E(258) and the A‐ring OH, and between Q(261) and the C‐ring C‐12 hydroxypropyl. In summary, the present study represents one of the first attempts to construct a homology model of the CB1 cannabinoid receptor based upon the published bovine rhodopsin x‐ray crystal structure and to elucidate the putative ligand binding site for nonclassical cannabinoid agonists. We postulated sites of the CB1 receptor critical for the ligand interaction, including the hydrophobic pocket interacting with the key pharmacophoric moiety, the C3 side chain. More work is needed to delineate between two alternative (and possibly other) binding conformations of the nonclassical cannabinoid ligands within the CB1 receptor. The present study provides a consistent framework for further investigation of the CB1 receptor–ligand interaction and for the study of CB1 receptor activation. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 169–189, 2003
An autocatalytic reaction as a model for the kinetics of the aggregation of β‐amyloid
Authors: Sabaté Raimon , Gallardo Montserrat , Estelrich Joan
Published: May 2003

Alzheimer's disease is the commonest form of senile dementia, affecting almost 20 million people worldwide. This neurodegenerative disorder is characterized by amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, β‐amyloid. Fibrillation of β‐amyloid is a nucleation‐dependent polymerization process, which is controlled by two kinetics parameters: the nucleation rate and the elongation or growth rate. As the kinetics of fibrillation is strongly dependent on the presence of trace amounts of fibrils, we suggest that the aggregation of β‐amyloid is a model of autocatalytic reaction. A mathematical analysis, permitting quantitative monitoring of the kinetics of fibrillogenesis of β‐amyloid, nucleation, and elongation constants, is presented. The model was checked by applying it to the aggregation of the fragment 1–40 of the β‐amyloid. Understanding of these rate constants may facilitate the study of the effect of substances used for controlling fibril creation and growth. The disaggregating effect of dodecyl trimethylammonium bromide, a cationic surfactant, was easily quantified by means of the model. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71:190–195, 2003
Mechanism of bactericidal and fungicidal activities of textiles covalently modified with alkylated polyethylenimine
Authors: Lin Jian , Qiu Shuyi , Lewis Kim , Klibanov Alexander M.
Published: May 2003

Our previous studies have led to a novel “nonrelease” approach to making materials bactericidal by covalently attaching certain moderately hydrophobic polycations to their surfaces. In the present work, this strategy is extended beyond the heretofore‐used nonporous materials to include common woven textiles (cotton, wool, nylon, and polyester). Pieces of such cloths derivatized with N‐hexylated+methylated high‐molecular‐weight polyethylenimine (PEI) are strongly bactericidal against several airborne Gram‐positive and Gram‐negative bacteria. In contrast, the immobilized and N‐alkylated PEIs of low molecular weight have only a weak, if any, bactericidal activity. These findings support a mechanism of the antibacterial action whereby high‐molecular‐weight and hydrophobic polycationic chains penetrate bacterial cell membranes/walls and fatally damage them. The bactericidal textiles prepared herein are lethal not only to pathogenic bacteria but to fungi as well. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 168–172, 2003.
Identification and comparison of aerobic and denitrifying polyphosphate‐accumulating organisms
Authors: Zeng Raymond J. , Saunders Aaron M. , Yuan Zhiguo , Blackall Linda L. , Keller Jürg
Published: May 2003

Two laboratory‐scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic–aerobic or alternating anaerobic–anoxic modes, respectively. Polyphosphate‐accumulating organisms (PAOs) were enriched in the anaerobic–aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic–aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well‐known PAO, “Candidatus Accumulibacter phosphatis” was abundant in both SBRs, and post‐FISH chemical staining with 4,6‐diamidino‐2‐phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic–anoxic SBR enriched for DPAOs was converted to anaerobic–aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic–aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5‐h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus‐uptake lag phase was not observed in the subsequent anaerobic–aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic–aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic–anoxic conditions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 140–148, 2003.
Protein loading, elution, and resolution behavior in a novel device that integrates ultrafiltration and chromatographic separation
Authors: Dai Xiao‐Ping , Majumdar Sudipto , Luo Robert G. , Sirkar Kamalesh K.
Published: May 2003

Hollow fiber membranes and chromatographic resin beads are commonly employed in a variety of bioseparation processes. A new class of integrated separation devices is being studied in which the shell side of a hollow fiber device is filled with adsorbents/chromatographic resin beads. Such devices and the corresponding separation methods integrate feed broth clarification by the microfiltration/ultrafiltration membrane with bioproduct purification by the shell‐side resin beads either as an adsorbent or as beads in elution chromatography. A mathematical model has been developed for the prediction of the chromatographic behavior of such an integrated device. Simulations have been done to study the effects of axial dispersion, feed flow rate, water permeation rate, fiber packing density, and void fraction. Numerical solutions were obtained by solving the governing equations. This model can reasonably describe the concentration profiles as well as the breakthrough and elution behaviors in the integrated device. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 125–139, 2003.
Biomass/adsorbent electrostatic interactions in expanded bed adsorption: A zeta potential study
Authors: Lin Dong‐Qiang , Brixius Peter J. , Hubbuch Jürgen J. , Thömmes Jörg , Kula Maria‐Regina
Published: May 2003

Expanded bed adsorption is an integrated technology that allows the introduction of particle‐containing feedstock without the risk of blocking the bed. The biomass particles contained in the feedstock have to be treated as an integral part of the process and potential interactions between suspended biomass and the adsorbent must be excluded during process design. Because the electrostatic forces dominate the interactions between the biomass and adsorbent, the zeta potential has been studied as a tool to characterize biomass/adsorbent electrostatic interactions. The zeta potentials of four types of biomass (yeast intact cells, yeast homogenate, Escherichia coli intact cells, and E. coli homogenate) and two types of ion exchanger were measured systematically at varying process conditions. Using the cell transmission index from biomass pulse‐response experiments as a parameter, the relations between zeta potential and the biomass/adsorbent interaction were evaluated. Combining the influences from zeta potential of adsorbent (ζa), zeta potential of biomass (ζb), and biomass size (d), parameter (−ζaζbd) was found to be an appropriate indicator of the biomass/adsorbent interactions in expanded beds under various liquid‐phase conditions for different types of biomass. The threshold value of parameter (−ζaζbd) can be defined as 120 mV2 μm for cell transmission of >90%, which means that systems with (−ζaζbd) < 120 may have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 149–157, 2003.
Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types
Authors: Kelm Jens M. , Timmins Nicholas E. , Brown Catherine J. , Fussenegger Martin , Nielsen Lars K.
Published: May 2003

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well‐rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF‐7, composed spheroids, revealed highly organized, three‐dimensional, tissue‐like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 173–180, 2003.
Cadmium tolerance and antioxidative defenses in hairy roots of the cadmium hyperaccumulator, Thlaspi caerulescens
Authors: Boominathan Rengasamy , Doran Pauline M.
Published: May 2003

Plant species capable of hyperaccumulating heavy metals are of considerable interest for phytoremediation and phytomining. This work aims to identify the role of antioxidative metabolism in heavy metal tolerance in the Cd hyperaccumulator, Thlaspi caerulescens. Hairy roots of T. caerulescens and the non‐hyperaccumu‐ lator, Nicotiana tabacum (tobacco), were used to test the effects of high Cd environments. In the absence of Cd, endogenous activities of catalase were two to three orders of magnitude higher in T. caerulescens than in N. tabacum. T. caerulescens roots also contained significantly higher endogenous superoxide dismutase activity and glutathione concentrations. Exposure to 20 ppm (178 μM) Cd prevented growth of N. tabacum roots and increased hydrogen peroxide (H2O2) levels by a factor of five relative to cultures without Cd. In contrast, growth was maintained in T. caerulescens, and H2O2 concentrations were controlled to low, nontoxic levels in association with a strong catalase induction response. Treatment of roots with the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), exacerbated H2O2 accumulation in Cd‐treated N. tabacum, but had a relatively minor effect on H2O2 levels and did not reduce Cd tolerance in T. caerulescens. Lipid peroxidation was increased by Cd treatment in both the hyperaccumulator and non‐hyperaccumulator roots. This work demonstrates that metal‐induced oxidative stress occurs in hyperaccumulator tissues even though growth is unaffected by the presence of heavy metals. It also suggests that superior antioxidative defenses, particularly catalase activity, may play an important role in the hyperaccumulator phenotype of T. caerulescens. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 158–167, 2003.
Separation of α‐lactalbumin and β‐lactoglobulin using membrane ultrafiltration
Authors: Cheang Beelin , Zydney Andrew L.
Published: May 2003

There is considerable commercial interest in the preparation of individual whey proteins as high‐value food additives, nutraceuticals, and therapeutics. This study examined the use of membrane filtration for the separation of α‐lactalbumin and β‐lactoglobulin. Stirred cell filtration experiments were performed using both cellulosic and polyethersulfone membranes to determine the optimal pH, ionic strength, and filtration conditions. Selectivities of greater than 55 could be achieved at pH 5.5 and 50 mM ionic strength using a 30‐kD cellulose membrane. A diafiltration process was then designed for the protein separation. A 16‐diavolume filtration yielded β‐lactoglobulin as the retentate product with a purification factor of 100 and recovery of 90%. The α‐lactalbumin was recovered in the filtrate with a purification factor of more than 10 and nearly 99% yield. Model calculations were in good agreement with the experimental data. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 201–209, 2003.
Constitutive versus thermoinducible expression of heterologous proteins in Escherichia coli based on strong PR,PL promoters from phage lambda
Authors: Menart Viktor , Jevševar Simona , Vilar Mateja , Trobiš Andreja , Pavko Aleksander
Published: May 2003

Constitutive and thermoinducible expression plasmids based on strong PR,PL promoters from phage lambda were compared for production of TNF‐alpha and its analogs under various conditions. Much higher accumulation of TNF was obtained in a constitutive system, so the wider applicability of such systems was studied. In constitutive systems, proteolytically susceptible proteins can be produced easily at low cultivation temperatures and the addition of expensive or toxic chemical inducers is not required. On the other hand, toxic proteins cannot be produced and selection pressure must be strictly maintained to ensure segregational stability of plasmids. Accumulation of TNF‐alpha and various analogs at levels up to 25% of total soluble protein was repeatedly achieved, which was 2–3‐fold higher than in a thermoinducible system. The stable behavior of the constitutive system in laboratory fermentors was also confirmed. We propose the constitutive system described here as a general model for many currently used expression systems containing strong but not completely repressed promoters. Such systems may be considered as constitutive ones with reduced promoter strengths, but still exhibiting all the intrinsic properties of constitutive expression systems. Although all modern expression systems are inducible, wider use of a constitutive system is evidently possible. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 181–190, 2003.
Asymmetric synthesis of L‐homophenylalanine by equilibrium‐shift using recombinant aromatic L‐amino acid transaminase
Authors: Cho Byung‐Kwan , Seo Joo‐Hyun , Kang Tae‐Won , Kim Byung‐Gee
Published: May 2003

L‐Homophenylalanine (L‐HPA) was asymmetrically synthesized from 2‐oxo‐4‐phenylbutyric acid (2‐OPBA) and L‐aspartate using a recombinant aromatic amino acid transaminase (AroAT). To screen microorganisms having such an L‐specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L‐HPA as a sole nitrogen source. To reduce the intracellular background synthetic activity by amino acid pools in the cells, a two‐step screening method was used. The putative AroAT (i.e., AroATEs) from the screened Enterobacter sp. BK2K‐1 was cloned, sequenced, and overexpressed in E. coli cells. The activity of the overexpressed AroATEs was 314‐fold higher than that of the wild‐type cell. The substrate specificities of the enzyme and homology search revealed that the cloned transaminase is true AroAT. The AroATEs showed a substrate inhibition by 2‐OPBA from 40 mM in the asymmetric synthesis, which made it difficult to perform batch asymmetric synthesis of L‐HPA at high concentrations of 2‐OPBA. To avoid the substrate inhibition by 2‐OPBA, intermittent addition of the solid‐state substrate was attempted to obtain a high concentration of L‐HPA. By using the cell extract (75 U) obtained from the recombinant E. coli harboring the AroATEs gene, the asymmetric synthesis of L‐HPA at 840 mM of 2‐OPBA resulted in >94% of conversion yield and >99% ee of L‐HPA of optical purity. Due to the low solubility (<2 mM) of L‐HPA in the reaction buffer, synthesized L‐HPA was continuously precipitated in the reaction media, which drives the reaction equilibrium towards the product formation. After full completion of the reaction, L‐HPA of high purity (>99% ee) was easily recovered by simple pH shift of the reaction media. This method can permit very efficient asymmetric synthesis of other unnatural amino acids using a single transaminase reaction. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 226–234, 2003.
Modulation of translation‐initiation in CHO‐K1 cells by rapamycin‐induced heterodimerization of engineered eIF4G fusion proteins
Authors: Schlatter Stefan , Senn Claudia , Fussenegger Martin
Published: May 2003

Translation‐initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine‐tunes the flow of information from gene to protein. In eukaryotes, translation‐initiation is typically initiated at a 7‐methyl‐guanylic acid cap posttranscriptionally linked to the 5′ end of mRNAs. Alternative cap‐independent translation‐initiation involves 5′ untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation‐initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap‐binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4GΔ and ΔeIF4G to redirect the cellular translation machinery exclusively to its own IRES‐containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap‐ and IRES‐dependent transgene mRNAs in mammalian cells. eIF4GΔ and ΔeIF4G were fused C‐ and N‐terminally to the FK506‐binding protein (FKBP) and the FKBP‐rapamycin‐binding domain (FRB) of the human FKBP‐rapamycin‐associated protein (FRAP), respectively. Rapamycin‐induced heterodimerization of eIF4GΔ‐FKBP and FRB‐ΔeIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose‐dependent manner. Rigorous quantitative expression analysis of cap‐ and IRES‐dependent SEAP‐ (human placental secreted alkaline phosphatase) and luc‐ (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization‐induced heterologous eIF4G in Chinese hamster ovary (CHO‐K1) cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210–225, 2003.
Fermentation of corn stover to carboxylic acids
Authors: Thanakoses Piyarat , Black Amanda S. , Holtzapple Mark T.
Published: May 2003

This article describes countercurrent fermentation to anaerobically convert corn stover and pig manure to mixed carboxylic acids using a mixed culture of mesophilic microorganisms. Corn stover was pretreated with lime to increase digestibility. The Continuum Particle Distribution Model (CPDM) was used to simulate continuous fermentors based on data collected from batch experiments. This model saves considerable time in determining optimum operating conditions. For 80% corn stover/20% pig manure, the highest total carboxylic acid productivity was 1.81 g/(L of liquid · d) at a concentration of 21.4 g total acid/L. The highest total acid selectivity, yield, and conversion were 0.714 g total acid/g volatile solids (VS) digested, 0.550 g total acid/g VS fed, and 0.770 g VS digested/g VS fed, respectively, at a concentration of 16.0 g total acid/L. CPDM predicted the acid concentration and conversion within 13.4 and 11.6%, respectively. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 191–200, 2003.
Vibrating membrane filtration for recovery and concentration of insect killing nematodes
Authors: Wilson J. A. , Postlethwaite J. , Pearce J. D. , Leach G. , Lye G. J. , Ayazi Shamlou P.
Published: May 2003

New experimental data are reported that demonstrate the use of a novel vibrating membrane filter (VMF) for the combined recovery and concentration of two species of nematodes, S. feltiae and P. hermaphrodita, from mature liquid fermentation cultures. The disk membrane module had a working surface area of 0.2 m2 and was operated at a constant flow rate of 0.2 m3 h−1. The recovery of the viable nematodes from the spent media and nonviable nematodes was assisted by an independently imposed oscillatory motion of the disk assembly, which produced an intense shear field at the membrane surface with calculated mean values on the order of 104 s−1. Adult (nonviable) nematodes in the fermentation culture were preferentially dissolved in a detergent (sodium dodecylsulfate) and successfully separated from the juveniles using the VMF equipment. Permeate fluxes on the order of 15 to 30 L/m2/h were achieved for an operating transmembrane pressure of 800 mbar. Industrial‐scale liquid fermentation for the manufacture of nematodes as biopesticides produces the viable nematode life stages in low‐concentration suspension containing large quantities of spent media and other waste material. The VMF equipment provided a flexible operation for separation, cleaning, and concentration of viable nematodes from the fermentation broths. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 235–240, 2003.
Ex vivo monitoring of protein production in baculovirus‐infected Trichoplusia ni larvae with a GFP‐specific optical probe
Authors: Kramer Shannon F. , Kostov Yordan , Rao Govind , Bentley William E.
Published: May 2003

Trichoplusia ni larvae were infected with baculoviruses containing genes for the expression of ultraviolet optimized green fluorescent protein (GFPuv) and several product proteins. A GFP‐specific optical probe was used to both excite the green fluorescent protein (λex = 385 nm), and subsequently monitor fluorescence emission (λem = 514 nm) from outside the infected larvae. The probe's photodetector was connected to a voltmeter, which was used to quantify the amount of GFPuv expressed in infected larvae. Voltage readings were significantly higher for infected vs. uninfected larvae and, by Western analysis, linear with the amount of GFPuv produced. In addition, the probe sensitivity and range were sufficient to delineate infection efficiency and recombinant protein production for model proteins, chloramphenicol acetyltransferase and human interleukin‐2. This work represents a critical step in developing an automated process for the production of recombinant proteins in insect larvae. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 241–247, 2003.
Neural networks teaching using Evenet‐2000
Authors: Gonzalez E. J. , Hamilton A. , Moreno L. , Aguilar R. M. , Marichal R. L.
Published: May 2003

In optimization problems, neural networks have been proved as an efficient method. Because of this, students of Engineering should learn this tool properly in subjects as “Optimal Control” or “Intelligent Control.” In this paper, a planning for neural networks teaching is proposed. As tool, Evenet‐2000, a Java‐based toolkit which allows to design and train neural networks with arbitrary architecture, is used. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 1–5, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10033
Digital control systems simulation using spreadsheets
Authors: El‐Hajj Ali , Karaki Sami , Kabalan Karim Y.
Published: May 2003

This study presents a method for simulating digital control systems using spreadsheets. This method consists of simulating as basic blocks: a delay, a first order transfer function, and a second order transfer function. Any transfer function in the z domain can be obtained by connecting a number of these blocks with appropriate coefficients. This simulation allows the determination of the time response of a sampled data system without solving equations. The simulation interface is customized using graphics and toolbars. The method is suitable for educational environments. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 6–12, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10034
MATLABbased GUIs for linear controller design via convex optimization
Authors: Khaisongkram Wathanyoo , Banjerdpongchai David
Published: May 2003

Owing to the current evolution of computational tools, a complicated parameter optimization problem could be effectively solved by a computer. In this paper, a CAD tool for multi‐objective controller design based on MATLABprogram is developed. In addition, we construct simple GUIs (using GUIDE tools within MATLAB) to provide a visual approach in specifying the constraints. The linear controller design problem can be cast as the convex optimization subjected to time domain and frequency domain constraints. This optimization problem is efficiently solved within a finite dimensional subspace by a practical ellipsoid algorithm. In the design process, we include a model reduction of the resulting controller to speed up the computational efficiency. Finally, a numerical example shows the capability of the program to design multi‐objective controller for a one‐link flexible robot arm. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 13–24, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10035
Development of a web‐based mass transfer processes laboratory: System development and implementation
Authors: Li Yusong , LeBoeuf Eugene J. , Basu P. K. , Turner Louis Hampton
Published: May 2003

A web‐based environment is utilized as a means to introduce advanced mass transfer processes concepts in environmental engineering and science courses. System development and implementation is presented, including detailed descriptions of the techniques employed to link software written in high‐level computer languages such as C++ and FORTRAN to an internet‐based, user‐friendly graphical user interface for both program input and output. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 25–39, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10036
Animated instructional media for stress transformations in a Mechanics of Materials course
Authors: Philpot Timothy A. , Hall Richard H. , Hubing Nancy , Flori Ralph , Oglesby David B. , Vikas Yellamraju
Published: May 2003

Two unique instructional modules—the Plane Stress Transformations Module and the Mohr's Circle for Plane Stress Module—developed for the Mechanics of Materials course are presented. Formative assessment data gathered from Mechanics of Materials students pertaining to these courseware modules are presented and discussed. General conclusions and recommendations are presented. © 2003 Wiley Periodicals, Inc. Comput Appl Eng Educ 11: 40–52, 2003; Published online in Wiley InterScience (www.interscience.wiley.com); DOI 10.1002/cae.10037
When training and prevention meet
Authors: Murphy Michael , Hall Olwen , Simpson Margaret
Published: May 2003

This article explores the intended and unintended consequences of CiN/child protection training when this training is offered to parents and grandparents who live in the community. It asks whether training can fulfil a preventative function on behalf of children and child protection systems. Copyright © 2003 John Wiley & Sons, Ltd.